Coimmunoprecipitation assays were performed as previously described. Briefly, cell lysates were incubated with antibody at 4 °C overnight. Antibody-bound complexes were precipitated with protein A + G agarose beads (Beyotime) and eluted by rinsing buffer, then the agarose-associated protein complexes were dissolved in SDS loading buffer and analyzed by immunoblotting assays.
Sample protein extraction and concentration determination of whole cells were performed as previously described [9 (link)]. Briefly, equal amounts of protein were run on SDS polyacrylamide gels and transferred to nitrocellulose membrane. The resulting blots were blocked with 5 % non-fat dry milk and probed with antibodies. The following antibodies were used: GAPDH (KangChen), MICAL1 (proteintech) (Santa Cruz Biotechnology), RAB35 (BD Biosciences) (ABclonal Technology), Akt, P-Akt, HA and GFP antibodies (Cell Signaling). Protein bands were detected by incubating with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized with ECL reagent (Millipore).
Sample protein extraction and concentration determination of whole cells were performed as previously described [9 (link)]. Briefly, equal amounts of protein were run on SDS polyacrylamide gels and transferred to nitrocellulose membrane. The resulting blots were blocked with 5 % non-fat dry milk and probed with antibodies. The following antibodies were used: GAPDH (KangChen), MICAL1 (proteintech) (Santa Cruz Biotechnology), RAB35 (BD Biosciences) (ABclonal Technology), Akt, P-Akt, HA and GFP antibodies (Cell Signaling). Protein bands were detected by incubating with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized with ECL reagent (Millipore).
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