Sumo1

Ubiquitination and SUMO assays were performed as described previously (31 (link), 65 (link)). At 36 h after transfection, HEK293T cells were treated for 12 h with 1 μM, 5 μM, and 10 μM of the proteasome inhibitor MG132. In addition, HEK293T cells transfected with wildtype or HCN4-R666Q mutant plasmid were treated with 5 μM MG132 for 12 h. Total protein was collected and analyzed by immunoblotting using the following antibodies: SUMO1 (1:1000, Proteintech) and SUMO2/3/4 (1:1000, Santa Cruz).

Total proteins were extracted from cells or tissues using the M-PER mammalian protein extraction reagent (Thermo Pierce, Rockford, USA) and measured using the BCA Protein Assay (Thermo Pierce). Protein samples (50 μg per assay) were separated on SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% skim milk and then incubated overnight at 4 °C in the presence of primary antibodies. The primary antibodies for p21, p53, KHSRP, c-Myc, EPCAM, and β-actin were purchased from Cell Signaling Technologies (CST, Danvers, MA, USA), p-c-Myc antibody was from Santa Cruz (CA, USA), and IκBα, Rb1, STAT3, histone-H3, SUMO1, and SUMO2/3 antibodies were from the Proteintech Company (Chicago, IL, USA). The membranes were washed 3 times in Tris-buffered saline with Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibody (CST, MA, USA) for 1 h at room temperature (RT). After washing 3 times with 15 mL of TBST, Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, USA) was added, and the bands were imaged with the Chemidoc XRS⁺ electrophoretic imaging system (Bio-Rad, Hercules, CA, USA). Density scanning of each protein band was performed using Image Lab software (Bio-Rad, Hercules, CA, USA).

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.