2.1. Cell culture and transfection
HEK 293 and HEK 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and transfected with FuGene 6 (Roche) according to the manufacturer’s recommendation. HeLa cells were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS, sodium pyruvate (1 mM) and transfected with FuGene HD (Roche) following the manufacturer’s protocol. The above media also contained penicillin (100 U/ml), streptomycin (100 mg/ml) and glutamine (2 mM).
2.2. Construction of Human serine/threonine phosphatase Expression Library
Human serine/threonine phosphatase clones were purchased from ATCC and Open Biosystem. Full-length cDNA sequence for each phosphatase containing an open reading frame was subcloned into pcDNA3.1 expression vector (Invitrogen). Mammalian PPP1CC expression vector was kindly provided by Dr. Sergei Nekhai (Howard University, Washington DC), PPM1D was obtained from Dr. Larry Donehower (Baylor College of Medicine, TX), and PPP5C from Dr. Xiaofan Wang (Duke University, NC).
2.3. Expression plasmids and small hairpin RNA expression constructs
The full-length open reading frame of the wildtype human PPM1A and PPM1B were subcloned in frame into mammalian expression vector pcDNA3.1 with an N-terminal 3Myc tag (Invitrogen). The PPM1A (R174G) and PPM1B (R179G) mutant expression constructs were generated by site-directed PCR mutagenesis (Stratagene) and verified by DNA sequencing. Mammalian expression vector for HA-IKKβ was obtained from Dr. Paul Chiao (The University of Texas MD Anderson Cancer Center, TX). The NF-κB-dependent firefly luciferase reporter plasmid and pCMV promoter-dependent Renilla luciferase reporter plasmid were purchased from Clontech (Mountain View, California). For bacterial expression of both PPM1A and PPM1B proteins, cDNAs encoding the wildtype (GST-PPM1A-wt and GST-PPM1B-wt) and phosphatase-deficient mutant version (GST-PPM1A-R174G and GST-PPM1B-R179G) of these two proteins were subcloned into pGEX-KG vector (Invitrogen) to generate glutathione S-transferase (GST) fusion proteins. A pSuper-retro vector (Ambion) was used to generate shRNA plasmids for PPM1A and PPM1B. For PPM1A, the following target sequences have been selected: 5′-AAGAGGAATGTTATTGAAGCC-3′ (shPPM1A-1), 5′-AAGTACCTGGAATGCAGAGTA-3′ (shPPM1A-2); and for PPM1B, target sequences were 5′-AATGCAGGAAAGCCATACTGA-3′ (shPPM1B-1), 5′-AACTTCTGGAGGAGATGCTGA-3′ (shPPM1B-2); pSuper-shRNA-control is: 5′-CTGGCATCGGTGTGGATGA-3′. The authenticity of these plasmids was confirmed by sequencing.
2.4. Antibodies and reagents
Antibodies against HA epitope, Myc epitope, NF-κB-p65, PCNA (PC-10) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-β-actin antibody was from Sigma-Aldrich Co. (St. Louis, MO). Antibodies against Phospho-IKKα/β and IKKβ were from Cell Signaling Technology, Inc. (Danvers, MA). Antibody against PPM1A was from Abcam Inc. (Cambridge, MA), and antibody against PPM1B was from Bethyl Laboratories, Inc. (Montgomery, TX). Recombinant human TNFα was purchased from the R & D Systems (Minneapolis, MN). FuGene 6 and FuGene HD transfection reagents were from Roche (Alameda, CA). Cell culture media were obtained from Invitrogen (Carlsbad, CA). Nitrocellulose membrane was obtained from Bio-Rad (Hercules, CA).
2.5. Luciferase reporter gene assays
The luciferase reporter gene assay was performed using a dual luciferase reporter assay system (Promega, Madison, WI) and a Monolight 3010 luminometer (BD PharMingen, San Diego, CA) as described previously [26
]. Briefly, targeted cells were transiently cotransfected with specific vectors and an NF-κB-dependent firefly
luciferase reporter construct as well as a Renilla
luciferase control construct. Cellular extracts were prepared 36 hrs post-transfection and the luciferase activities were determined. Relative NF-κB luciferase activity was normalized to Renilla
luciferase activity. Data are presented as the mean ± standard deviation and are representative of three independent experiments.
2.6. Quantitative reverse transcription PCR (qRT-PCR) analyse
Total RNAs were prepared using TriZol reagent (Invitrogen) from HeLa pSuper-shRNA-control, pSuper-shPPM1A and pSuper-shPPM1B cells. qRT-PCR was carried out by using 100 ng of total RNA. A volume of 10 μl of 2x QuantiTect SYBR Green RT-PCR Master Mix (Qiagen), 0.2 μl QuantiTect RT Mix (Qiagen), 1 μl of 10 μM forward and reverse primers, and 6.8 μl of RNase-Free Water were added to each sample for analysis by absolute quantification. qRT-PCR was performed in 96-well plates with the DNA Engine Opticon™ System (MJ Research). The mRNA levels of target genes in the samples were normalized against β-actin. Each target gene was measured in triplicate. The primers were designed by using the Primer3.0 software and are as follows: IL-6: 5′-CACACAGACAGCCACTCACC-3′ and 5′-TTTTCTGCCAGTGCCTCTTT-3′; β-actin: 5′-ACCGCGAGAAGATGACCCAG-3′ and 5′-TTAATGTCACGCACGATTTCCC-3′. Human IL-6 expression in different HeLa stable cell lines was also analyzed by RT-PCR. In this assay, and cDNA was prepared from the total RNA isolated with TriZol Reagent, using SuperScript III Gene Expression Tools (Invitrogen) according to the manufacturer’s protocol. PCR was performed on 1 μl aliquots from each cDNA reaction, using human IL-6 and β-actin primer sets (IL-6, 30 cycles; β-actin, 20 cycles). The PCR products were subjected to electrophoresis on a 2% agarose gel.
2.7. Generation of stable HeLa cells expressing shRNA targeting PPM1A or PPM1B
The pSuper-PPM1A or PPM1B retroviral construct was transfected into HEK 293T cells with retrovirus packing vector Pegpam 3e and RDF vector using FuGene 6 transfection reagent. Viral supernatants were collected after 48 and 72 hours. HeLa cells were incubated with virus-containing medium in the presence of 4 mg/ml polybrene (Sigma Aldrich). Stable cell lines were established after 5 days of puromycin (2 μg/ml) selection and knockdown of the target gene was confirmed by Western blotting.
2.8. Preparation of nuclear and cytosolic fractions
Nuclear and cytosolic extracts were made as described [27
]. In brief, cells were harvested in ice-cold PBS (pH 7.4) and were pelleted by 500 ×g for 3 min and then lysed for 30 min on ice in buffer B (10 mM HEPES buffer, pH 7.9, containing 0.1 mM EDTA, 10 mM KCl, 0.4% (v/v) IGEPAL, 0.5 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF)). Cell lysates were centrifuged at 15,000×g for 15 min, 4°C. The resulting supernatants constituted cytosolic fractions. The pellets were washed three times with buffer B and then resuspended in buffer C (20 mM HEPES buffer, pH 7.9, containing 400 mM NaCl, 1 mM EDTA, 1 mM DTT and 1 mM PMSF) and incubated for 30 min on ice, then centrifuged at 15,000×g for 15 min. The supernatants were used as nuclear extracts.
2.9. Immunoblotting and immunoprecipitation
Cells were harvested in ice-cold PBS (pH 7.4) and spun down. The pellets were dissolved in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% IGEPAL, 0.25% Na-deoxycholate, 1 mM PMSF, 1 mM DTT, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM Benzamidine, 20 mM disodium p-nitrophenylphosphate (pNPP), 0.1 mM sodium orthovanadate (OV), 10 mM sodium fluoride (NaF), phosphatase inhibitor cocktail A and B (Sigma Aldrich)). The cell lysates were either subjected directly to 10% SDS-PAGE for immunoblotting analysis or immunoprecipitated for 3 hrs with the indicated antibodies. Immune complexes were recovered with protein A/G-agarose (Santa Cruz Biotechnology) for 3 hrs, then washed three times with wash buffer containing 20 mM HEPES (pH 7.4), 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, and 0.05% Triton X-100. For immunoblotting, the immunoprecipitates or 10% whole cell lysates (WCL) were resolved on SDS-PAGE and transferred to nitrocellulose membranes. The membranes were immunoblotted with various antibodies, and the bound antibodies were visualized with horseradish peroxidase-conjugated antibodies against rabbit or mouse IgG using the ECL-Plus Western blotting system (GE Healthcare Biosciences Corp., USA) according to the manufacturer’s instruction.
2.10. Purification of GST-PPM1A and PPM1B fusion proteins
All the above-mentioned GST plasmids (GST-PPM1A-wt, GST-PPM1A-R174G, GST-PPM1B-wt and GST-PPM1B-R179G) were transformed into E. coli BL-21 strain (Invitrogen), and then the bacteria were grown in Luria broth at 37°C to an A600=0.6 before induction with 0.1 mM isopropyl β-d-thiogalactoside (IPTG) for 4 hrs at 30°C. Bacteria were pelleted and lysed with extraction buffer (50 mM Tris–HCl, pH 8.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 50 mg/ml lysozyme, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM PMSF) 45 min on ice. The bacteria were sonicated at 4°C in 1% Sarcosyl (Sigma Aldrich), after which Triton X-100 (1%), 5 μg/ml DNase, and 5 μg/ml RNase (Roche) were added. The lysates were centrifuged at 15,000×g and the supernatants containing GST fusion proteins were collected. Fusion proteins were purified from cell lysates using glutathione–sepharose beads (Sigma Aldrich) overnight at 4°C. The beads were washed three times in extraction buffer containing 0.5% Triton X-100, one time in extraction buffer containing 0.1% Triton X-100. Proteins were eluted in elution buffer (30.7% glutathione, 50 mM Tris-HCl, pH 8.0, 20% glycerol, 5M NaCl) and dialyzed in PBS. The protein concentrations were then assessed with a Bradford Protein Assay (Bio-Rad). The proteins were visualized by 10% SDS-PAGE and Coomassie blue staining of the gel.
2.11. Phosphatase Assays
HEK 293T cells seeded onto 10 cm dishes were transfected with the HA-IKKβ expression plasmid. The HA-IKKβ proteins were immunoprecipitated from cell extracts with anti-HA antibody. After washing beads three times with the wash buffer, the immunoprecipitated HA-IKKβ were then incubated with or without recombinant GST-PPM1A or GST-PPM1B wildtype or phosphatase-deficient mutant proteins in phosphatase 2C buffer (250 mM imidazole, pH 7.2, 1 mM EGTA, 25 mM MgCl2, 0.1% 2-mercaptoethanol, 10% BSA) or Lambda Protein Phosphatase (λ-Ppase) at 30°C for 30 min. The phosphatase reactions were then terminated by boiling in protein sample buffer and proteins were separated by 10% SDS-PAGE. The levels of HA-IKKβ phosphorylation were measured by immunoblotting analysis with antibody against phospho-IKKβ.