Anti β actin

Total proteins were extracted from transfected cells using RIPA lysis buffer containing PMSF (Solarbio) and protease inhibitor cocktail (Promega) according to the instructions of the manufacturer. The protein concentration was determined by BCA Protein Assay Kit (Multi Sciences). After mixed with loading buffer and heated at 99°C for 5 minutes, the protein lysates were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Millipore). The transferred membranes were blocked with 5% skim milk for 1 hour at room temperature and incubated overnight with the specific primary antibodies at 4°C. Subsequently, the membranes were incubated at room temperature with horseradish peroxidase–conjugated goat anti‐rabbit IgG (KPL) for 1 hour. The bands were visualized with enhanced chemiluminescence (ECL) detection reagent (Multi Sciences) by Chemi XT 4 (Syngene). The primary antibodies were used: anti‐β‐actin (AC026, ABclonal), anti‐E‐cadherin (E‐AB‐35932, Elabscience), anti‐N‐cadherin (E‐AB‐64011, Elabscience) and anti‐FAM83H (ab121816, Abcam).

The proteins from skin fibroblasts were separated by the sodium dodecyl sulphate- (SDS-) polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene fluoride (PVDF, Millipore, Billerica, MA, USA) membrane. The membranes were blocked by 5% milk without fat for 2 h, then incubated with anti-α-SMA (1 : 2000; Bosterbio, Wuhan, China), anti-PCNA (1 : 2000; ABclonal, Wuhan, China), anti-receptor interacting protein kinase 1 (RIPK1), RIPK3, anti-mixed lineage kinase domain like protein (MLKL), p-MLKL (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH (1 : 3000; Sigma-Aldrich, St. Louis, MO, USA), anti-β-tubulin (1 : 3000; CMCTAG, Milwaukee, WI, USA), or anti-β-actin (1 : 5000; ABclonal, Wuhan, China) antibodies at 4°C overnight. Next, the secondary antibody (1 : 5000; Beyotime, Shanghai, china) was incubated for 2 h at room temperature. The protein bands were visualized on the membrane with enhanced chemiluminescence (ECL, Thermo Fisher Scientific Inc., Rockford, IL, USA) solution.

Cells or skin tissues pretreated with the homogenate were lysed into lysates in RIPA Lysis Buffer (cat. P0013C, Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (cat. B14002, B15002, Bimake, USA). Lysates (20 μg protein/lane) were separated by SDS-PAGE and transferred to PVDF membranes (cat. IPVH00010, Millipore, USA). After blocking for 1 h at room temperature with 3% BSA in TBST, the PVDF membranes were incubated with specific primary antibodies for overnight at 4 °C. The membranes were washed with TBST at least 3 times and then incubated with appropriate secondary antibody at room temperature. Anti-G9A (cat. ab185050, Abcam, UK, 1:1000), anti-Caspase3 (cat. 9662, CST, USA, 1:1000), anti-BCL-2 (cat. 12789-1-AP, Proteintech, Wuhan, China, 1:2000), anti-BAX (cat. 50599-2-Ig, Proteintech, 1:2000), anti-GAPDH (cat. 60004-I-Ig, Proteintech, 1:3000), anti-P65 (cat. 8242 S, Abcam, 1:1000), anti-Pho-P65 (cat. 3033 S, CST, 1:1000), anti-NF-κB1 (cat. 13586 S, CST, 1:1000), anti-TRAF6 (cat. ab33915, Abcam, 1:2000), anti-EDAR (cat. ab137021, Abcam, 1:1000), anti-EDARADD (cat. A15950, Abclonal, Wuhan, China, 1:1000), anti-β-ACTIN (cat. sc-47778, Santa cruz, USA, 1:500), anti-TUBULIN (cat. 11224-1-AP, Proteintech, 1:2000), HRP Goat Anti-Mouse IgG (H + L) (cat.AS003, Abclonal), HRP Goat Anti-Rabbit IgG (H + L) (cat. AS014, Abclonal) were used as antibodies.

Total protein of fifteen midgut tissues obtained from larvae was extracted by using Protein Kit (OMEGA Bio-tek, GA, USA). The amount of total protein from midgut samples was quantified using a BCA kit (Solarbio Life Sciences, Beigjing, China). Then 35 μg protein for each treatment were resolved on SDS-PAGE gels and electrotransferred to PVDF filters. After blocking in 1% non-protein blocking solution (Sangon Biotech, Shanghai, China) for 40 min at RT, the PVDF filters were incubated for 4 h with one of the following primary antibodies anti-β-actin (ABclonal Technology, Wuhan, China) at 1: 30000 dilution, anti-Histone H3 (ABclonal Technology, Wuhan, China) at 1: 3000 dilution and anti-Phospho-Histone H3 (ABclonal Technology, Wuhan, China) at 1:1000 dilution in PBS containing 0.1% Tween-20 (PBST). After washing with PBST three times for 10 min each, the PVDF filters were incubated with corresponding secondary Goat anti-rabbit antibody labelled with FITC (Solarbio Life Sciences, Beigjing, China) in PBST at 1:1000 dilution for 1 h at RT. Images were obtained with a Typhoon 9410 scanner (GE Healthcare, USA) and analyzed with Image J software.

The total proteins of the cells were lysed in a RIPA buffer containing 1% protease inhibitors (Beyotime, Shanghai, China) and collected. After SDS-PAGE, the proteins were transferred onto PVDF membranes. The primary antibodies were anti-Cav1.2 (Alomone, Jerusalem, Israel), anti-Nav1.5 (Alomone, Jerusalem, Israel), anti-Kv4.2 (Abcam, Cambridge, England), anti-HOXa10 (ABclonal, Wuhan, China), and anti-β-Actin (ABclonal, Wuhan, China). The HRP-conjugated secondary antibody HRP-labeled goat anti-rabbit IgG (Absin, Shanghai, China) was used.

Western blotting was performed as previously described (15 (link)). The primary antibodies were used at the following dilutions: anti-PTIP (1:1000, Bethyl), anti-CtIP (1:1000, Proteintech), anti-CtIP (1:500, Santa Cruz), anti-RPA32-S33 (1:1000, Bethyl), anti-β-actin (1:3000, Abclonal), anti-FALG (1:2000, Invitrogen), anti-HA (1: 1000, Proteintech), anti-UFL1 (1:1000, Bethyl), anti-GST (1:1000, Abclonal), and anti-His (1:1000, Abclonal).