Anti his

Two purified recombinant human glutathione S‐transferase (GST)‐tagged SMARCA4 segments [amino acids (aa) 612–656 and (aa) 1455–1566] (GeneCreate Biological Engineering Co., Ltd., Wuhan, China) and His‐tagged human NOTCH3 intracellular domain (aa 1665–2321) (GeneCreate Biological Engineering Co., Ltd.) were mixed and incubated on an ice bath for 3 h. Subsequently, the mixture was incubated with Streptavidin Magnetic Beads (New England Biolabs, Lpswich, MA, USA). After washing five times with the washing buffer, the proteins were eluted with the wash buffer supplemented with 15 mm of reduced glutathione. The elutes were separated using 12% SDS/PAGE and transferred onto the PVDF membranes (Millipore, Billerica, MA, USA), and then finally probed with anti‐GST (Proteintech) and anti‐His (Proteintech) antibodies. GST from Wuhan Genecreate (Wuhan, China) was used as the negative control.

Cells were harvested in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% deoxycholate, and a protease inhibitor cocktail (Roche, Basel, Switzerland)). The lysates were then subjected to 12% SDS-PAGE and Western blotting according to our standard procedures [18 (link),44 (link)]. The antibodies used in this study included anti-tubulin (1:3000), anti-His (1:10,000), anti-Flag (1:5000), and anti-Dicer (1:2000), which were purchased from Proteintech, Rosemont, IL, USA. The JAK1/JAK2 inhibitor Ruxolitinib (INCB018424) was purchased from Selleck, Houston, TX, USA (CAS No. 941678-49-5).

The following antibodies were purchased: anti-Flag (MBL, M185-3L), anti-HA (MBL, M180-3), anti-VAPB (Proteintech, 14477-1-AP and 66191-1-Ig), anti-SCRN1 (Proteintech, 14303-1-AP), anti-GAPDH (Proteintech, 60004-1-Ig and 10494-1-AP), anti-poly/mono-ADP-ribose (Cell Signaling Technology, 83732S), PAR/pADPr (R&D Systems, 4335-MC-100), anti-Calnexin (Novus, NB300-518), anti-His (Proteintech, 66005-1-Ig), anti-GST (Proteintech, 66001-2-Ig), DyLight 488-conjugated goat anti-mouse IgG (Abbkine, A23210), Dylight 594-conjugated goat anti-rabbit IgG (Abbkine, A23420), horseradish peroxidase (HRP)-conjugated mouse anti-rabbit IgG LCS (Abbkine, A25022), HRP-conjugated goat anti-mouse IgG (Proteintech, 15014), and HRP-conjugated goat anti-rabbit IgG (Proteintech, 15015).
Antibodies against ARTC1, ARTC3, ARTC4, and ARTC5 were generated by immunizing rabbits with recombinant proteins GST-ARTC1 (N23–C295), GST-ARTC3 (N27–C362), GST-ARTC4 (N47–C285), and GST-ARTC5 (N23–C291), respectively, purified in our laboratory. Serum samples were obtained from immunized rabbits and incubated with AminoLink Plus Coupling Resin (Thermo, 20501) in Pierce Disposable Plastic Columns (Thermo, 29922). The resin was washed three times with PBS. Antibodies were eluted by 100 mM Glycine (pH 2.5).

The plasmid vector encoding V5‐tagged α1‐antitrypsin, influenza virus HA, bovine preprolactin, and cVIMP‐cys were gifted from Lisa Swanton (University of Manchester), Mary Jane Gething (University of Melbourne), Stephen High (University of Manchester), and Lars Ellgaard (University of Copenhagen), respectively. The β1‐integrin, hemagglutinin, and the soluble Sec‐Cys variant of SelS/VIMP (cVIMP‐cys) sequences have been described previously (Gething et al, 1980; Tiwari et al, 2011; Christensen et al, 2012). Auranofin, carmustine, and DHEA were purchased from Sigma‐Aldrich; TRi2 synthesis will be described elsewhere (Stafford, W. et al, manuscript in preparation). The commercial antibodies used were anti‐His (Proteintech), anti‐V5‐tag (Invitrogen), and 9EG7 (BD Bioscience). Antibody to LDLr (121) was a gift from Ineke Braakman (Utrecht University; Pena et al, 2010). Purified recombinant ChaC1 enzyme was a gift from David Ron (University of Cambridge; Tsunoda et al, 2014). The plasmid containing human thioredoxin (hTrx) with a streptavidin binding peptide (SBP) tag was a gift from Tobias Dick (German Cancer Research Centre, Heidelberg). Recombinant Trx protein was purified as described previously (Schwertassek et al, 2007).

Yeast protein was extracted from intact cells using Yeast Total Protein Extraction kit (Sangon Biotech, China); protein concentrations were determined by the Bradford method (Bradford, 1976 (link)). Fifty microgram samples were separated by SDS-PAGE and used for western blot analysis. Western blots were performed as previously described (Cao et al., 1999 (link)). The primary antisera were anti-His (Proteintech, United States) and anti-Actin (Proteintech, United States) diluted 1:2,000. The secondary antibody was goat anti-mouse IgG-alkaline phosphatase conjugate (Proteintech, United States) diluted 1:3,000.

Tissues or cultured cells were lysed with RIPA lysis buffer with protease inhibitors and phosphatase inhibitors (Beyotime, Shanghai, China) using standard methods. After centrifugation and quantification, 25 µg of protein was loaded for blotting with the indicated specific antibodies. A NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific, Waltham, USA) was used to isolate nuclear and cytoplasmic fractions according to the manufacturer’s protocol. The following antibodies were used: anti-Sox9 (1:1000, Abcam, Cambridge, UK), anti-HNF4α (1:2000, Abcam, Cambridge, UK), anti-TERT (1:1000, Novus, Colorado, USA), anti-E-Cadherin (1:1000, Abcam, Cambridge, UK), anti-Vimentin (1:1000, CST, Danvers, USA), anti-CK19 (1:1000, Abcam, Cambridge, UK), anti-LGR5 (1:1000, Abcam, Cambridge, UK), anti-Epcam (1:1000, Abcam, Cambridge, UK), anti-P65 (1:1000, CST, Danvers, USA), anti-p-P65 (1:1000, CST, Danvers, USA), anti-Bcl3 (1:100, Abcam, Cambridge, UK), anti-Histone H3 (1:1000, Proteintech, Rosemont, USA), anti-YAP1 (1:1000, CST, Danvers, USA), anti-CTGF (1:1000, Abcam, Cambridge, UK), anti-Cyr61 (1:1000, Abcam, Cambridge, UK), anti-His (1:5000, Proteintech, Rosemont, USA), anti-Flag (1:2000, Proteintech, Rosemont, USA), anti-Myc (1:1000, Proteintech, Rosemont, USA), anti-Ubiquitin (1:1000, CST, Danvers, USA), and anti-β-actin (1:4000, Bioworld, MN, USA).