Anti pkd3

Western Blotting was performed according to standard procedures. Whole-cell extracts (50–100 μg) prepared using IP-lysis buffer containing 10 mM Tris HCl, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, and 1% Triton-X100 supplemented with Complete Protease Inhibitor Cocktail Tablets (Roche) were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred to PVDF membranes (Millipore Corp., USA). Membranes were blocked using 5% dry milk in PBS containing 0.2% Tween 20. For subsequent washes, 0.2% Tween 20 in PBS was used. Membranes were incubated with primary antibodies over-night at 4 °C under shaking conditions. The following primary antibodies were used: anti-PKD2 (Bethyl, #A300-073 A), anti-PKD3 (Bethyl, #A300-319A), anti-PKD1 (Santa Cruz, #sc-935), anti-phospho PKD (Ser744/748) (Cell Signalling, #2054), anti-PKCδ (Santa Cruz, #sc-213) and anti-phospho PKCδ (Thr 505) (Cell Signalling, #9374). This was followed by incubation with secondary horseradish peroxidase (HRP)–coupled antibodies diluted 1:3000, 1 h at RT. Detection was performed with either ECL or ECL+ kits (Thermo scientific, USA).

After BCR stimulation or PMA treatment, total B cell contents were extracted using 1% Nonidet P-40 lysis buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.5 mM EDTA, supplemented with proteases inhibitors (Sigma-Aldrich). Proteins were separated on SDS-PAGE and analysed by western blotting with the appropriate antibodies: anti-phospho-Erk1/2, anti-PKD1/2, anti-phospho-Ser^744/748 PKD, anti-phospho- Ser⁹¹⁶ PKD, anti-HSP90 (Cell Signaling Technology), anti-PKD2 (Millipore, Molsheim, France), anti-PKD3 (Bethyl Laboratories Inc, Souffelweyersheim, France), anti-CXCR4 (Abcam, Paris, France), anti-phospho-Ser³³⁹ CXCR4 (Abcam), anti-phospho-Ser^324/325 CXCR4 (ECM Biosciences, Souffelweyersheim, France) and anti-α-Tubulin (Sigma-Aldrich). Detection was achieved using chemi-luminescence (ECL, GE Heathcare, Velizy-Villacoublay, France) and visualized using the ChemiDoc MP Imaging System (BioRad, Marnes-la-Coquette, France).