Anti phospho eif2α

Mouse podocytes were incubated with various concentrations of Ang II (Sigma-Aldrich Inc., St. Louis, MO, USA) for 24 h. To inhibit phosphoinositide 3(PI3)-kinase/Akt signaling, cells were exposed to 5 μM LY294002 (Cell Signaling Technology, Danvers, MA, USA), a PI3-kinase inhibitor. For Ang II inhibition, losartan (Merck, MSD LTD., Seoul, Korea) was used in a concentration of 10⁻⁶ M for 24 h. The primary antibodies for Western blotting were purchased as follows: anti-phospho-PERK and anti-β-tubulin antibodies from Santa Cruz Biotechnology (CA, USA) and anti-Bip, anti-ATF4, anti-phospho-eIF2α, and anti-eIF2α antibodies from Cell Signaling (Beverly, MA, USA).

Agents were purchased/obtained as follows: (1) bortezomib (Biovision, Palo Alto, CA); (2) fumonisin B1, palmitate, 4-PBA (4-phenylbutyric acid), TUDCA (tauroursodeoxycholic acid), and anti-α-tubulin, anti-HA and anti-CerS2 antibodies (Sigma-Aldrich, St Louis, MO); (3) ceramides (fatty acyl lengths C16, C18, C20, C22, and C24) and C17 ceramide (d_17:1/C_18:0) (Avanti Polar Lipid, Alabaster, AL); (4) anti-GRP78, anti-CHOP, anti-FAS, anti-phospho-eiF2α, anti-eiF2α, anti-PERK, and anti-SCD-1 antibodies (Cell Signaling Technology, Beverly, MA); (5) anti-SREBP-1, anti-phospho-PERK and anti-CerS6 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); (6) anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) antibody (Millipore, Temecula, CA); (7) anti-INSIG-1 antibody (Abcam, Cambridge, MA); (8) anti-mouse-HRP (horseradish peroxidase) and anti-rabbit-HRP antibodies (Jackson Laboratory, Bar Harbor, ME).

Cells were collected using PBS and lysed for 10 min on ice using RIPA buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; Pierce, 89901) supplemented with proteinase inhibitor cocktail (Roche 1186153001) and PhosSTOP (Roche 04906845001). Samples were centrifuged for 20 min at 4°C at 14,000 rpm. 4X NuPAGE LDS sample buffer (Thermo Fisher Scientific NP0008) was added to the supernatant and samples were boiled for 5 min. Samples were run in 4–12% NuPAGE Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes using an iBlot 2 transfer device (Thermo Fisher Scientific). Membranes were blocked with Odyssey blocking buffer (LI-COR) and then incubated with primary antibodies. Following incubation with dye-labeled secondary antibodies, signals were visualized using an Odyssey Fc imaging system (LI-COR). Primary western blot antibodies were anti-β-actin (Santa Cruz Biotechnology sc-1616), anti-eIF2α (Santa Cruz Biotechnology sc-133132), anti-phospho-eIF2α (Cell Signaling 3597S), anti-mCherry (Abcam 167453), and anti-G3BP1 (BD biosciences 6111126). Secondary western blot antibodies were IRDye 800CW/680RD (LI-COR) used at a dilution of 1:15,000.