Total RNA was extracted from HeLa cells with Nucleospin RNAII (Machery-Nagel) and reverse-transcribed with Titanium One-Step RT-PCR kit (Clontech Laboratories, Inc.) according to the manufacturer's instructions.
Reverse transcription was performed using 1 μg of purified RNA, oligo dT, and SuperScript III reverse transcriptase (Invitrogen) in a 20-μl reaction.
Amplification of each specific transcript was performed using RT2 PCR primer Set (SuperArray). Plasmids containing cDNAs for each specific PKD isoform were used as standards for real-time quantitative PCR amplification (Q-PCR). These plasmids were 10-fold serially diluted and used as templates for the Q-PCR to generate standard curves (ranging from 102 to 105 copies/μl). Real-time Q-PCR assays were performed with an Mx4000 Multiplex Quantitative QPCR System (Stratagene). Reactions were performed using 200-nM primers, 1 μl template/25 μl PCR reaction and the iTaq SYBR Green Supermix with ROX (Bio-Rad). A two-step PCR method (denaturation at 95°C for 30 s and annealing/extension at 60°C for 1 min) was used. Each assay included the analysis of the samples in duplicates. In addition, samples were run at least three times to check for interassay variability. Melting curve analyses were performed on all PCR reactions to check for specificity of the amplification. Real-time qRT-PCR analyses for β-actin were included as housekeeping genes to normalize the data.
Antibodies in this study included goat anti-GST (GE Healthcare), rabbit anti-GST (AbCam), sheep anti–human TGN46 (AbD Serotec), rabbit affinity-purified anti-PKD3 (Bethyl), rabbit affinity-purified anti-PKD2 (Bethyl) monoclonal anti-β-actin (Sigma-Aldrich), monoclonal anti-Flag (Sigma-Aldrich), AMCA donkey anti–rabbit (Jackson ImmunoResearch Laboratories), and Texas red donkey anti–sheep (Jackson ImmunoResearch Laboratories). The monoclonal antibody 8G5F11, which recognizes the extracellular domain of VSV-G, was provided by Dr. Douglas Lyles (Wake Forest University School of Medicine, Winston-Salem, NC).
Cell culture and transfection
293-T cells, HeLa cells, and the cell line stably expressing a GFP-tagged form of mannosidase II (HeLa MannII-GFP) were grown in complete medium consisting of DME (Cellgro) containing 10% FCS and supplemented with 0.8 mg/ml of geneticin for HeLa MannII-GFP at 37°C in a 7% CO2 incubator. The cells were transfected with FuGene 6 (Roche) or Lipofectamine 2000 (Invitrogen) following the manufacturer's recommendations.
The day before transfection, HeLa cells were plated in order to ensure 50% confluency on the day of transfection. Knockdown transfections were performed using 80 nM of purified siRNA and Lipofectamine 2000 according to the manufacturer's protocol. For the VSV-G transport assay, specific targeting siRNA and siGlo Risc free-labeled nonspecific siRNA were mixed (ratio 4:1) with a final concentration of 100 nM. siRNAs controls were from Dharmacon. The siRNA PKD3 and the siRNA PKD2 are Silencer-validated siRNAs from Ambion.
ssHRP and PLAP secretion assay
30 h after transfection with siRNA, the cells were cotransfected with the SS-HRP-Flag and the pSEAP2-basic (Clontech Laboratories, Inc.) plasmid (carrying PLAP cDNA), using Lipofectamine 2000 (Invitrogen). 30 μl of extracellular media was harvested 48 h after the initial siRNA transfection. HRP activity was measured using enhanced chemiluminescence (ECL) as described previously (Bard et al., 2006
). PLAP activity in the medium and in the cells was measured using the Phospha-Light System (Applied Biosystems) following the manufacturer's protocol. PLAP activity inside the cells was normalized by total protein concentration and used to normalize both HRP and PLAP secreted into the medium.
VSV-G transport assay
30 h after transfection with siRNA, the cells were transfected with ts045VSV-G-GFP construct and cultured at 40°C for 20 h. 100 μg/ml of cycloheximide was then added before a 2-h incubation at 20°C. After an incubation at 32°C for 40 min the cells were harvested with a cell dissociation buffer (Invitrogen) and fixed with 4% paraformaldehyde. After blocking with PBS containing 1.5% serum and 0.1% sodium azide, the labeling of surface VSV-G–GFP was performed for a 30-min incubation at 4°C with the anti-VSV-G mAb 8G5F11, which is specific for the extracellular domain of VSV-G. After washings with the blocking buffer, the cells were incubated with the secondary (APC)-labeled anti–mouse IgG antibody (Jackson ImmunoResearch Laboratories) for 30 min at 4°C. After washing, the cells were analyzed on a FACScalibur flow cytometer (BD Biosciences). The amount of VSV-G present at the cell surface (APC positive) of cells transfected with both siRNA (cy3 positive) and VSV-G (GFP positive) after subtracting the background was normalized by the GFP intensity.
Transfected 293-T cells or HeLa cells were lysed in 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, and protease inhibitors for 30 min at 4°C. After centrifugation at 13,000 rpm for 10 min, protein concentrations were measured in the lysates. 100 μg of extracts were incubated with the primary antibody (1:100) at 4°C and after 2 h, 30 μl of G-Sepharose beads (GE Healthcare) were added for 1 h. Immobilized proteins were released by boiling in Laemmli buffer and analyzed by SDS-PAGE. For Flag-tagged proteins, 100 μg of cell lysates were incubated with anti-Flag M2 affinity gel and eluted with 3×Flag peptide following the manufacturer's instructions (Sigma-Aldrich).
In vitro kinase assay
An equal amount of the indicated proteins purified by immunoprecipitation from transfected 293-T cells was incubated for 10 min at 32°C in a buffer containing 50 mM Tris-HCl, 30 mM MgCl2, 0,3 mM ATP, 2 mM DTT, 0.2 μM PdBu, and 5 μCi ATPγP32. The reaction was stopped by addition of 6× SDS sample buffer and the samples were processed for SDS-PAGE and autoradiography.
In vitro binding assay
After immunoprecipitation with anti-GST antibody from transfected 293-T cell lysates, the purified GST-tagged proteins bound to the beads were incubated with equal amount of purified Flag-tagged proteins for 2 h at 4°C in 300 μl of PBS containing 0.1% Triton X-100 and 0.2%BSA. After extensive washes in the same buffer, the precipitates were eluted in 1× SDS sample buffer and processed for Western blotting.
PKD-CAAX constructs cloning
PKD-CAAX-CA, PKD-CAAX-WT, and PKD-CAAX-KD were cloned by PCR using GST-PKD-CA, GST-PKD-WT, and GST-PKD-KD, respectively, as templates as previously described (Maeda et al., 2001
). The CAAX motif was appended by using a reverse primer containing the nucleotides coding for the CAAX motif MVLC: 5′-AAATCTAGAAAGCTTTCACATAACGAGACAGAGGATGCTGACACGCTCACTG-3′.
24 h after transfection, HeLa cells expressing MannII-GFP grown on coverslips were fixed with 4% formaldehyde in PBS for 10 min, blocked, and permeabilized with blocking buffer (0.05% Saponin and 0.2% BSA in PBS) for 20 min. The coverslips were incubated with primary antibodies diluted in blocking buffer for 2 h, washed, incubated with secondary antibodies diluted in blocking buffer for 1 h, washed, mounted using Fluor Save Reagent (Calbiochem), and visualized with a Nikon Eclipse TE2000-U microscope. Pictures were taken using MetaMorph software and deconvolved using AutoVisualize+AutoDeblur 9.3 software. The pictures were then opened in ImageJ v1.37 and Adobe Photoshop.
HeLa cells were depleted of both PKD2 and PKD3 as described above. The cells were transfected with SS-HRP and processed for immunoelectron microscopy, and HRP was visualized by staining with DAB and H2
as described previously (Polishchuk et al., 2000
). HeLa cells were transfected with GST-tagged PKD WT, PKD-CAAX-CA, PKD-CAAX-WT, or PKD-CAAX-KD. Cells were fixed in the mixture of 4% paraformaldehyde and 0.5% glutaraldehyde, washed, and labeled with anti-TGN46–specific antibody followed by an antibody conjugated with peroxidase as described previously (Polishchuk et al., 2000
). Then cells were incubated with polyclonal antibody against GST and subsequently with Nanogold-conjugated Fab fragment of anti–rabbit IgG. Nanogold particles were enhanced using the manufacturer's kit (Nanoprobes). ST-HRP-expressing cells were incubated directly with DAB and H2
(Polishchuk et al., 2000
) and then labeled with anti-GST antibody as described above. Cells were then embedded in Epon 812 and thin sections visualized in a Tecnai-12 electron microscope (FEI, Philips). Images were taken using an Ultra View CCD digital camera. Morphometric analysis of Golgi stacks was performed in 20 cells for each experimental condition using the ANALYSIS software.
Tubular profiles were defined as HRP-positive structures with length twice or more higher than thickness.
The statistical significance of the difference between means was determined using the t test. Differences were considered significant at P < 0.01.