Secondary hrp linked antibodies

HeLa cells were obtained from ATCC and maintained in DMEM with 10% FBS. Anti-GST antibody was from Santa Cruz (Santa Cruz, CA), anti-HA, anti-FLAG (M2) and anti-β-actin from Sigma-Aldrich (St Louis, MO), anti-GFP/YFP (Ab290) from Abcam (Cambridge, MA), anti-PKD3 and anti-SSH1L from Bethyl Laboratories (Montgomery, TX), anti-pS744/748-PKD (recognizes pS706/710 for human PKD2 and pS731/735 for human PKD3), anti-LIMK, anti-pT508-LIMK, anti-PAK4, anti-pS474-PAK4, anti-Cofilin and anti-pS3-Cofilin from Cell Signaling Technology (Danvers, MA), anti-PKD2 from Millipore (Billerica, MA). The monoclonal antibodies directed against PKD1 and pS978-SSH1L were described and characterized previously [20] (link). Secondary HRP-linked antibodies were from Millipore. Secondary antibodies for immunofluorescence analysis were goat-anti-mouse Alexa Fluor 488 F(ab’)2 and goat-anti-rabbit Alexa Fluor 488 F(ab’)2 from Invitrogen (Carlsbad, CA). TransIT HeLa-Monster (Mirus, Madison, WI) was used for transient transfection of cells.

All antibodies used for immunoprecipitation, immunoblotting, immunofluorescence or immunohisto-chemistry are described in detail in Table 1. The phosphorylation-specific anti-pS650-PIP5K1C antibody was a gift from Dr. De Camilli and is described in [8 (link)]. The anti-pS448-PIP5K1C antibody was made by 21^st Century Biochemicals (Marlboro, MA, USA) and is further described in [9 (link)]. Ac-NTVFRKN[pS]SLKSSPSK-Ahx-C-amide and C-Ahx-SNTVFRKN[pS]SLKSSPS-amide were used as blocking peptides for this antibody. Secondary HRP-linked antibodies were from Millipore (Billerica, MA, USA) and secondary antibodies for immunofluorescence (Alexa Fluor 488 F(ab’)2 fragment of goat-anti-rabbit IgG or Alexa Fluor 568 F(ab’)2 fragment of goat-anti-mouse) were from Invitrogen (Grand Island, NY). 12-Phorbol 13-myristate acetate (PMA) was from Sigma (St. Lois, MO, USA). Expression plasmids for HA-tagged PIP5K1C, as well as the expression constructs for tagged constitutively-active versions (S to E mutations in critical activation loop serines) of PKD1, PKD2 or PKD3 have been described in detail elsewhere [9 (link)]. Lipofectamine 2000 (Invitrogen) was used for transient transfections.