Gtx109639

Cells were trypsinized, pelleted, and washed twice in PBS. Cells were lysed by heating for 5 min in SDS sample buffer (62.5 mM Tris-HCl (pH = 6.8), 1% (v/v) 2-mercaptoethanol, 1% (w/v) sodium dodecyl sulfate, SDS, 10% (w/v) glycerol, and 0.02% (w/v) bromophenol blue, BPB). Lysates were resolved on 7.5–10% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 30 min in blocking buffer (5% bovine serum albumin (Sigma-Aldrich) in T-TBS buffer (0.05% Tween 20 (Sigma-Aldrich) in TBS (tris-buffered saline))) and subsequently incubated with the primary antibodies in T-TBS buffer overnight at 4 °C. The antibody for LC3B (2775) was purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The antibody for detection of β-actin (GTX109639) was purchased from GeneTex (Irvine, CA, USA). The antibody for GAPDH (ab8245) was purchased from Abcam (Cambridge, UK). For the quantification of signals, all samples to be compared were run on the same gel. Bands were quantified using ImageJ^{31 ,32 (link)}. All bands to be compared were quantified on the same image and were within the linear range of detection of the software.

The primary antibodies used are listed as follows: anti-cytochrome c (1:1,000, 10993-1-AP, Proteintech, rosemont, IL, USA), anti-caspase-9 (1:1,000, #9508, Cell Signaling, Danvers, MA, USA), anti-PERK (1:1,000, #3192, Cell Signaling, Danvers, MA, USA), anti-phosphorylated-PERK (anti-p-PERK; Thr982;1:800, DF7576, Affinity Biosciences, Cincinnati, OH, USA), anti-IRE1 (1:500, ab37073, Abcam, Cambridge, MA, USA), anti-p-IRE1 (phosphor S724; 1:1,000, ab48187, Abcam, Cambridge, MA, USA), anti-activating transcription factor 6 (anti-ATF6; 1:1,000, ab203119, Abcam, Cambridge, MA, USA), anti-N’ATF6 (1:800, ab37149, Abcam, Cambridge, MA, USA) and anti-β-actin (1:3,000, GTX109639, GeneTex, Irvine, CA, USA). The ER stress activator Tunicamycin (TM) and the p-PERK inhibitor GSK2656157 were obtained from MedChem Express (Monmouth Junction, NJ, USA). The p-IRE1 inhibitor 3,5-dibromosalicylaldehyde (DBSA) was obtained from Tokyo Chemical Industry (TCI, Tokyo, Japan).

The cells were exposed to different treatments for the indicated time, and the cells were lysed on ice with lysis buffer. The cell lysate was cleared by centrifugation at 12,000 g for 10 min. The lysate was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were transferred to a polyvinylidene fluoride membrane. After blocking with 5% non-fat dry milk in Tris-buffered saline, the membrane was incubated overnight with the desired primary antibody (FPN (Novus Biologicals, NBP1-21502, 1:1000), FTH-1 (Cell Signaling, 4393S, 1:1000), GPX4 (Abcam, ab125066, 1:1000), TFRC (Cell Signaling, 13208s, 1:1000), xCT (Cell Signaling, 17681s, 1:1000), GAPDH (GeneTex, GTX100118, 1:10000), α-SMA (Abcam, ab5694, 1:1000), fibronectin (Santa Cruz, sc-9068, 1:1000), N-cadherin (BD, 610920, 1:1000), and β-actin (GeneTex, GTX109639, 1:10000). Subsequently, the membrane was incubated with an appropriate secondary antibody. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) method and captured by a luminescence imaging system (LAS 4000™, Fuji Photo Film Co., Ltd.).

Whole proteins were extracted and resolved by SDS–PAGE, and transferred to a polyvinylidene difluoride membrane. The membrane was blocked in 5% nonfat milk at room temperature for 1 h, followed by incubation with primary antibody at 4  °C overnight. Primary antibodies were used to detect HA (Roche, 12CA5, 1:1,000), Myc (Roche, 9E10, 1:1,000), β-actin (GeneTex, GTX109639, 1:1,000), Cip1 pT65 (1:500), and Cip1 pT73 (1:500). Signals were developed using Luminata^TM Crescendo Western HRP Substrate (Millipore). The image was quantified by ImageJ software. All uncropped western blots can be found in Supplementary Fig. 11.

The BACH1 protein was detected by Western blotting using either the mouse anti-BACH1 monoclonal antibody 9D11 (1:500, generated in-house) or rabbit anti-BACH1 antiserum A1–6 (1:1000, generated in-house) as reported previously [18 (link),53 (link)]. Other antibodies used in the experiments were as follows: rabbit anti-ACTB (1:1000, GTX109639, GeneTeX, Inc. Irvine, CA, USA), rabbit anti-TBK1 (1:1000, D1B4, Cell Signaling, Danvers, MA, USA), mouse anti-SRC (1:1000, 130124, Santa Cruz Biotech, Santa Cruz, CA, USA), rabbit anti-FBXO22 (1:1000, 13606-1-AP, Proteintech, Rosemont, IL, USA), mouse anti-DDDDK-tag (1:1000, M185-3L, MBL Life Science, Woburn, MA, USA), rabbit anti-His-tag (1:1000, PM032, MBL Life Science, Nagoya, Japan) and rabbit anti-Ubiquitin (1:1000, MFK-003, Nippon Bio-Test Laboratories Inc., Tokyo, Japan). HRP-conjugated anti-rabbit IgG (1:2500, NA934V, GE Healthcare, Fairfield, CT, USA) and HRP-conjugated anti-mouse IgG (1:2500, NA931V, GE Healthcare, Fairfield, CT, USA) were used as secondary antibodies.

Aorta tissues were homogenized in radioimmunoprecipitation assay (RIPA; Solarbio, R0010, Beijing) lysis buffer. Equal amounts of protein (40 μg) were extracted from the total aorta and separated by 10% SDS–polyacrylamide gel electrophoresis (Solarbio, P1200, Beijing). The proteins were then transferred to polyvinylidene fluoride (PVDF; Millipore, ISEQ00010, USA) membranes and blocked with 5% skim milk powder (BD, 232100). The following primary antibodies were incubated with PVDF membranes at 4°C overnight: NF-κB antibody (1 : 2000, GeneTex, GTX102090), anti-thioredoxin interacting protein (TXNIP) antibody (EPR14774) (1 : 1000, Abcam, ab188865), NLRP3 antibody (1 : 1000, GeneTex, GTX64347), anti-ASC antibody (1 : 200, Abcam, ab175449), caspase-1 antibody (14F468) (1 : 1000, GeneTex, GTX14367), IL-1β antibody (1 : 2000, GeneTex, GTX55675), and β-actin antibody (1 : 5000, GeneTex, GTX109639). The PVDF membranes were then extensively rinsed and incubated at room temperature with secondary antibodies (1 : 5000, Proteintech, SA00001-1, SA00001-2, Wuhan) for 1 h. A biomolecular imager (Fuji, LAS-4000, MINI, Japan) and QualityOne were used to scan and quantitate the membranes. All experiments were repeated 3 times.