Secondary hrp conjugated goat anti rabbit igg

Immunoblot analysis was carried out to evaluate the accumulation of PKR protein. The procedure was published previously [31 (link)]. Briefly, total cells lysates were prepared from cells by SDS sample buffer 1X [62.5 mM Tris-HCl (Tris(hydroxymethyl)aminomethane hydrochloride) pH 6.8; 50 mM DTT (dithio-threitol); 10% glycerol; 2% SDS (sodium dodecyl sulfate); 0.01% Bromophenol Blue; EDTA-free Protease Inhibitor Cocktail 1X (Roche, Basel, Switzerland)], sonicated, and boiled for 5 min. An equal amount of protein extracts was subjected to SDS-gel electrophoresis (SDS-PAGE), transferred to membranes (Bio-Rad Life Science Research, Hercules, CA, USA), and subjected to immunoblot analysis. Specific bands were visualized using Immobilon Classico Western HRP substrate (Merk, Millipore, Rahway, NJ, USA). Anti-GAPDH (sc-32233) and PKR (A12) (sc393038) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and secondary HRP-conjugated goat anti-rabbit IgG was purchased from Millipore.

Anti-p-CKR-5(E11/19), which specifically recognizes phosphoserine at position 349 (a specific GRK phosphorylation site) [12 (link)], anti-CKR-5 (D6), anti-GAPDH MAbs, anti-p-ERK1/2 (Thr 202/Tyr 204), anti-ERK1 (C-16), and Rab5 (FL-215) polyclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). β-Arrestin1/2 (D24H9) rabbit monoclonal antibody, which detects endogenous level of total β-arrestin1 and -2 proteins, shows a single band at 50 KDa, as reported in the datasheet, was purchased from Cell Signaling Technology (Beverly, MA, USA). Secondary HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Millipore; secondary FITC-conjugated goat anti-mouse IgG was from ICN Biomedicals (Aurora, OH, USA). Secondary HRP-conjugated anti-rabbit and anti-mouse IgG VeriBlot for IP were from Abcam (Cambridge, UK).