Sumo2 3

Antibodies used in this study are listed below where respective dilutions are mentioned in the parentheses:
α-actinin, mouse monoclonal, Sigma-Aldrich (1:400); SUMO2/3, rabbit monoclonal, Cell signaling (1:1000); SUMO2 + 3, mouse monoclonal, Abcam (1:1000); Calcineurin A, mouse monoclonal, BD Bioscience (1:250); GAPDH, mouse monoclonal, Sigma-Aldrich (1:20,000); Histone H3, rabbit polyclonal, Cell-signaling (1:1000); α-Tubulin, mouse monoclonal, Sigma-Aldrich (1:8000); Ubiquitin, mouse monoclonal, Millipore (1:1000); STAT1, rabbit monoclonal, Cell signaling (1:1000); p-STAT1, rabbit monoclonal, Cell signaling (1:1000); STAT3, rabbit monoclonal, Cell signaling (1:1000); p-STAT3, rabbit monoclonal, Cell signaling (1:1000); F4/80, rat monoclonal, Dianova (1:500); HA, mouse monoclonal, Sigma-Aldrich (1:20,000); HectD3, rabbit polyclonal, Mybiosource (1:1000); V5, mouse monoclonal, Biozol (1:1000).

Lysate, IP or His‐PD Samples were resolved by SDS–PAGE (10–15% gels) and transferred to Immobilon‐P membranes (Millipore Inc.), which were then immunoblotted with the following antibodies against: β‐actin (Sigma), CHIP (Cell Signaling), Drp1 (Cell Signaling), Fis1 (Proteintech), Flag (Proteintech), FTH1 (Cell Signaling), GAPDH (Santa Cruz biotechnology), GST (GE Healthcare), LC3 (Cell Signaling), p62 (Cell Signaling), SENP3 (Cell Signaling), SENP5 (Proteintech), SUMO‐1 (Santa Cruz biotechnology), SUMO‐2/3 (Cell Signaling; MBL) or Tom20 (Santa Cruz biotechnology). Immune complexes were detected using either HRP‐conjugated secondary antibodies (Sigma) followed by enhanced chemiluminescence (GE Healthcare) or using fluorescent secondary antibodies (LI‐COR).
Each immunoblot presented is representative of at least three experiments carried out using different cell populations.

Primary VSMCs were homogenized and lysed in RIPA buffer supplemented with protease inhibitor tablets (Roche). Protein was separated (10 μg, for each sample) on gradient (4–12%) SDS-PAGE, followed by semi-dry transfer onto PVDF membranes (Millipore, Bedford, MA). Membranes were immunoblotted with primary antibody and the appropriate horseradish peroxidase-conjugated secondary antibody (Bio-Rad). Immunoblots were analyzed by enhanced chemiluminescence technique (Thermo Fisher). Primary antibodies to IRE1α, TG2, GAPDH, SUMO1, SUMO2/3, HA, and K48 ubiquitin were from Cell Signaling Technology (Beverly, MA).

Transfected cells were washed with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails. The supernatant was collected and preserved at –80 °C. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes, and incubated with 5% skim milk for 1 h. The membranes were incubated overnight at 4 °C with the following specific primary antibodies: human DAXX (diluted 1:200), SUMO-2/3 (Cell Signaling Technology (CST), Danvers, MA, USA; #4971), α-Tubulin (CST; #3873), Lamin A/C (CST; #4777), HA-Tag (CST; #3724) (diluted 1:1000), and GAPDH (diluted 1:1000, Proteintech, Chicago, USA; 60004-1-Ig). The membranes were then incubated with secondary horseradish peroxidase (HRP)-conjugated antibodies (dilution 1:5000, Cell Signaling Technology) for 2 h at 25 °C. Proteins were then detected using enhanced chemiluminescence reagent and observed using a Biorad Imaging System (Biorad). The grayscale value represents the amount of target protein and was calculated by gray scanning using ImageJ software (NIH, Bethesda, USA). All protein expression levels were evaluated relative to GAPDH expression.

The above prepared cell lysates or immunoprecipitates were separated on 10% (vol/vol) polyacrylamide gels and transferred onto PVDF membranes. The lysates and imminoprecipitates were probed with primary antibodies, including SUMO1, SUMO2/3 (Cell Signaling, Danvers, MA, USA), SUMO-specific protease 3 (SENP3), SUMO-specific protease 7 (SENP7) and beta-actin (Santa Cruz, Delaware, California, USA), and UBC9 (Cell Signaling, Danvers, MA, USA) for quantitative Western blot analysis, respectively, to confirm that the CSE insulted HBEs underwent a change in terms of total SUMOylation levels or patterns [14 (link)]. Similarly, to verify that CYP1A1 was SUMOylated by SUMO1, the above precipitates were probed with a CYP1A1 antibody (13241–1-AP) (Proteintech Group, Inc., Wuhan, China) and the reactive bands were developed using the established techniques [17 (link)]. Briefly, the membranes were incubated with an indicated primary antibody at 4 °C overnight. After washes with TBST (0.5% Tween) five times, the membranes were probed with an appropriate HRP-conjugated secondary antibody for 1 h, and the reactive bands were visualized using the ECL reagents (Servicebio, Wuhan, China) as instructed. Quantitative analysis of relative intensity of each band was conducted using the Image J software and β-actin was used for normalization.