Novex bis tris poly acrylamide gel

Proteins were resolved on 4%–12% Novex Bis-Tris poly-acrylamide gel (Invitrogen) and blotted onto nitrocellulose membrane (Invitrogen). Membranes were incubated with the following primary antibodies overnight, before the addition of HRP-conjugated secondary antibodies. P-P70S6K (T389), P70S6K, P-S6 (S240/244), S6, P-AKT (S473), AKT, P-AMPK (T172), AMPK, TSC2, P53 (1C12), Ubiquitin, P-H2AX, and GAPDH were from Cell Signaling Technology. HPRT-1, REDD1, SESN2, and MDM2 were from Proteintech. ZFP36L2 was obtained from Abcam, MilliporeSigma, and Santa Cruz Biotechnology. The presence of target protein was visualized using Super Surgical Western Pico ECL substrate (Pierce). Quantification of Western blotting images was done using ImageJ (NIH). Antibody suppliers and catalog numbers are shown in Supplemental Table 4.

Tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor (Thermo Fisher Scientific). Protein concentration in lysates was determined using BCA Protein Quantification Kit (Pierce) and as described previously (Sawicki et al., 2018 (link)). Equal amounts of proteins were resolved on 4–12% Novex Bis-Tris poly-acrylamide gel (Invitrogen) and blotted onto nitrocellulose membrane (Invitrogen). After blocking with tris-buffered saline containing 0.05% Tween 20 (Thermo Fisher Scientific) and 5% milk, the membrane was incubated overnight at 4°C in primary antibody against HPRT (ProteinTech), succinate dehydrogenase complex flavoprotein subunit A (SDHA, Invitrogen), FTN (Sigma-Aldrich), IRP2 (Novus Biologicals), TfR1 (Invitrogen), FPN1 (Novus Biologicals), Hepcidin (Abcam), Ubiquitin (Cell Signaling Technology), GAPDH (ProteinTech), and α-Tubulin (ProteinTech). The following day, membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Jackson ImmunoResearch) for 1 hr and proteins were visualized using Super Surgical Western Pico ECL substrate (Pierce). Quantification of immunoblotting image was done using ImageJ (NIH).

Proteins were resolved on 4–12% Novex Bis-Tris poly-acrylamide gel (Invitrogen) and blotted onto nitrocellulose membrane (Invitrogen). Approximately 20–40 μg of whole cell lysate and approximately 6–10 μg of cytosolic or mitochondrial fractions were loaded onto the gel. Membranes were incubated with primary antibodies for ABCB8 (made in-house), ABCB7, MIA40, ALR, Tim 23, 6x-His, and GFP (ProteinTech), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ATP synthase F1 subunit alpha (ATP5a), cytochrome oxidase subunit 4 (COX4) and α-tubulin (Abcam), FLAG (Sigma), β- actin (Cell Signaling), GPAT (a generous gift from Dr. Roland Lill [University of Marburg]), or 70 kDa subunit SDH (Invitrogen) overnight, before the addition of horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch). The presence of target protein was visualized using Super Surgical Western Pico ECL substrate (Pierce). Quantification of western blotting image was done using ImageJ (NIH).