Ha antibody

Total Akt1/2/3 antibody was from Santa Cruz Biotechnology, HA antibody was from Sigma; anti-phospho-Akt (S129) and its not-phosphorylated counterpart were raised and purified as elsewhere described [22 (link)]. Secondary antibodies towards rabbit and mouse IgG, conjugated to horse radish peroxidase, were from PerkinElmer.

Antibodies for Western blotting were as follows: CAPS antibody raised against the full-length protein in rabbit, purified by protein A–agarose chromatography, and used at 1:1,000; GAPDH antibody purchased from Life Technologies (catalogue number AM4300) and used at 1:4,000; Rbcn3α antibody purchased from AbCam (catalogue number ab234771) and used at 1:000; Rbcn3β antibody purchased from Sigma-Aldrich (catalogue number HPA042074) and used at 1:1000; V-ATPase V0A antibody purchased from Sigma-Aldrich (catalogue number HPA022144) and used at 1:1000; V-ATPase V1A antibody purchased from AbCam (catalogue number 137574) and used at 1:1000; GFP antibody purchased from Sigma-Aldrich (catalogue number G1544) and used at 1:2000; and HA antibody purchased from Sigma-Aldrich (catalogue number H3663) and used at 1:2000. Rbcn3β antibody for immunofluorescence was purchased from Protein Tech (catalogue number 24431-1-AP) and used at 1:50, and Rbcn3α antibody for immunofluorescence was obtained from Sigma-Aldrich (HPA039375) and used at 1:50.

E. coli cells overexpressing the constructs of interest were grown and lysed as described above. 50 µl of the cleared bacterial lysates (corresponding to 2 OD₆₀₀ units) were five times diluted in lysis buffer without DNase and RNase, and containing 0.5% Igepal CA630 (Sigma-Aldrich). Samples were pre-cleared by the adding 25 µl of 50% Sepharose CL-6B beads (Sigma-Aldrich) previously washed with a wash buffer containing 50 mM Na-Pi-buffer pH 8.0, 300 mM NaCl, 5% glycerol and 0.5% Igepal for 30 min at 4 °C (while rotating). The beads were pelleted and the supernatants were incubated overnight with 7 µg HA antibody (Sigma-Aldrich) or 10 µg mouse IgG antibody (Santa Cruz Biotechnology, Inc) at 4 °C. Protein G Sepharose 4 Fast flow beads (GE Healthcare Life Sciences) washed twice with wash buffer were added to each IP and incubation was continued for 2 h at 4 °C. Beads were pelleted, washed three times with wash buffer and once with 50 mM Na-Pi-buffer [pH 8.0] containing 150 mM NaCl, and bound proteins were eluted by incubating the beads for 10 min at 37 °C with Laemmli sample buffer (62.5 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 0.004% bromophenol blue) supplemented with 50 mM DTT. Approximately 40% of the eluates (corresponding to 0.8 OD₆₀₀ units) and ~ 10% of the total lysates (corresponding to 0.2 OD₆₀₀ units) were loaded on NuPAGE gels, and analyzed by immuno-blotting.

HEK293 cells were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) Fetal Bovine serum (GE Healthcare, Little Chalfont, UK), 2 mm glutaMAX (ThermoFisher Scientific, Waltham, MA, USA), 100U·mL⁻¹ Penicillin and 100 μg·mL⁻¹ Streptomycin (ThermoFisher Scientific). Anti‐GST, Anti‐FLAG M2 antibody, HA antibody and agarose resins were purchased from Sigma (St. Louis, MO, USA). Glutathione sepharose 4B beads were from GE healthcare. Anti‐phosphotyrosine antibody (4G10) was from Millipore (Billerica, MA, USA), PKD anti‐pSer‐744/748 antibody, anti‐PKCδ antibody, secondary HRP‐linked goat anti‐Rabbit and Horse, anti‐Mouse antibodies were from Cell Signaling Technologies (Beverly, MA, USA). An in‐house site‐specific phospho‐Tyr antibody targeting pTyr in the P + 1 loop (CPApYLAPEV), which is cross‐reactive between PKD isoforms due to 100% homology of the epitope is described previously 36. Phorbol 12,13‐dibutyrate (PDB), ATP, STI‐571, PP2, CRT 0066101, CID 755673 and Hydrogen peroxide 30% (v/v) were from Sigma, Polyethyleneimine (PEI) was from Polysciences Inc. (Warrington, PA, USA).

For Co-IP assays, A. tumefaciens N. benthamiana leaves were harvested to extract proteins with IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 5 mM DTT, protease inhibitor cocktail) as previously described^{55 (link)}. The leaf extracts were centrifuged to obtain a supernatant and incubated with GFP and Protein A beads (Invitrogen) for 6–8 h at 4 °C. The beads were recovered from the mixture by centrifugation and washing three times with cold washing buffer. Total protein was separated in 10% SDS-PAGE gel for Western Blot analysis by using an HA antibody (Sigma, Shanghai, China) and Pierce ECL western blot substrate (Thermo Fisher Scientific, Rockford, IL, USA).