Hrp conjugated β actin

Western blotting samples were prepared as previously described [9 (link)]. Following SDS-PAGE, the following primary antibodies were used: total ZIP7 (19429-1-AP, Proteintech^TM); pZIP7 (MABS1262, Merck Millipore); pAKT^S473 (No. 9271, Cell Signalling). All were used at 1:1,000 dilution. HRP-conjugated β-actin (A3854, Sigma Aldrich) used at 1:10,000 was utilized as loading control. Densitometry analysis was performed using Alpha DigiDoc software, with results being normalized to their respective loading control.

Proteins from an equivalent number of cells (50,000 cells) were separated by gel electrophoresis on 4–12% SDS gel and then transferred to nitrocellulose membrane. For immunoblots with the monoclonal anti-citrulline antibody (clone F95, mouse IgM, EMD Millipore) and HRP conjugated β-actin (Sigma-Aldrich), membranes were directly blocked with fish gelatin without chemical modification. Alternatively, membranes were chemically modified as previously described [12 (link)], blocked with milk, and detected with anti-modified citrulline antibody (AMC, rabbit polyclonal, kindly provided by Dr. Felipe Andrade, Johns Hopkins University). The primary antibodies were used as follows: anti-modified citrulline antibody (1 : 4,000, 4°C, overnight), monoclonal anti-citrulline F95 antibody (1 : 1,000, 4°C, overnight), and β-actin (1 : 40,000, 37°C, 1 h). For β-actin, enhanced chemiluminescence (ECL) was directly used for detection. For AMC and F95, HRP conjugated anti-rabbit IgG and anti-mouse IgM were, respectively, used as secondary antibody before ECL detection. Densitometric analysis was performed with ImageJ (NIH).