Ab51254

Mouse lung tissue and MLE-12 cells were lysed by RIPA lysate. Mouse lung tissue needs to be additionally put into a homogenizer to fully lyse. The protein lysate was then centrifuged (12,000 g, 15 min, 4°C) and the supernatant was collected. After being mixed into the loading buffer, the protein lysate was heated to 100°C for 5 min. Then, we made a 10% sodium dodecyl sulfate polyacrylamide gel and added 10 μL of protein lysate. After electrophoresis and membrane transfer, the protein is transferred to the polyvinylidene fluoride membrane. We used 5% bovine serum albumin-PBS-Tween to block nonspecific antigens for 1 h at room temperature. The membrane was then incubated with primary antibody dilutions (β-actin, ab8226, Abcam, Cambridge, MA, USA; IL-1β, ab254360, Abcam, Cambridge, MA, USA; tumor necrosis factor (TNF), ab1793, Abcam, Cambridge, MA, USA; SOD1, ab51254, Abcam, Cambridge, MA, USA; SOD2, ab68155, Abcam, Cambridge, MA, USA; caspase 3, ab184787, Abcam, Cambridge, MA, USA; p65, ab16502, Abcam, Cambridge, MA, USA; p-p65, ab86299, Abcam, Cambridge, MA, USA; IκBα, ab183503, Abcam, Cambridge, MA, USA;) at 4°C overnight. After washing the membrane, we incubated the membrane at room temperature for 1 h with the secondary antibody dilution (Abcam, Cambridge, MA, USA). Finally, we used chemiluminescent liquid for color development.

For the protein expression analysis of SOD1, Hmox1 and GCLc proteins, PNS treated INS-1 cells were washed with ice-cold phosphate buffer saline (PBS), collected and lysed using RIPA lysis buffer (P0013C, Beyotime, China) supplemented with a protease inhibitor PMSF (ST506, Beyotime, China) according to manufacturer’s protocol to obtain total protein. Equal amounts of protein lysates were resolved in 10% SDS-PAGE (SK6010-250, Coolaber, China), transferred on the Immobilon-PSQ PVDF blotting membrane (ISEQ00010, Merck) using wet transfer. Membranes were incubated in 5% BSA (ST023-200 g, Beyotime, China) in Tris-buffered saline pH 7.6 containing 0.1% Tween-20 following primary antibody incubation overnight. Antibodies used against SOD1 (dilution: 1:50000, ab51254), Hmox1 (dilution: 1:50000, ab68477), GCLc (dilution: 1:20000, ab190685) were purchased from Abcam. GAPDH (dilution: 1:100000, 60004-1-Ig) was purchased from Protein Tech. Membranes were then washed with TBST and incubated with secondary antibody Anti-Mouse IgG, HRP-linked antibody (A0208, Beyotime, China) and Anti-Rabbit IgG, HRP-linked antibody (A0216, Beyotime, China). Protein detection was performed using ECL western blotting substrate (SL1350, Coolaber) and captured with Amersham Imager 600 (GE Healthcare Life Sciences). Expression of GAPDH was used as a control.

Brucine, ferric ammonium citrate (FAC), and glutathione (GSH) were all purchased from Sigma-Aldrich (Saint Louis, MO, USA). Ferrostatin-1 (Fer-1), liproxastin-1, and 4-phenylbutyrate (4-PBA) were all obtained from Selleck Chemicals (Houston, TX, USA). Primary antibodies against GRP78 (ab12685), ATF3 (ab254268), ATF4 (ab184909), GPX4 (ab125066), cystine-glutamate antiporter xCT (ab175186), ferritin light chain (ab69090), ferritin heavy chain (ab75972), FPN (ab78066), TF (ab82411), TFR (ab1086), ATG5 (ab108327), LC3B (ab192890), p62 (ab109012), Beclin-1 (ab207612), superoxide dismutase 1 (SOD1) (ab51254), and catalase (ab209211) were all from Abcam (Cambridge, UK). Anti-PERK (#5683) and anti-β-Actin (#4970) antibodies were from Cell Signaling Technology (Beverly, MA, USA). The other reagents were purchased from Sigma-Aldrich.