Anti yap

Co-IP was performed as described previously30 (link). The reagents used were protein A/G-Sepharose (Novex, Oslo, Norway) and Western/IP lysis buffer (Beyotime, Haimen, China). The antibodies used for IP were anti-YAP (Santa Cruz Biotechnology, #sc101199) or anti-FLAG (CST, #8146).

For crude protein extracts, cells were lysed in RIPA buffer containing 1% NP40 and 0.1% SDS (Carl Roth) supplemented with ‘Complete’ and ‘PhosStop’ protease/phosphatase inhibitor cocktail (Sigma Aldrich) as described^{50 (link)}. Immunodetection of cellular extracts was performed using an anti-KIBRA (Santa Cruz Biotechnology; 1:500), anti-SP1 (Merck; 1:1000), anti-YAP (Santa Cruz Biotechnology; 1:1000), anti-pYAP (Ser127; Cell Signaling; 1:1000), anti-LATS1 (Merck; 1:1000), anti-pLATS1 (Thr1079; Cell Signaling; 1:500) and anti-rabbit secondary antibody (Santa Cruz Biotechnology; 1:20000 or Merck; 1:10000). Sample loading was controlled by β-actin detection (Cell Signaling; 1:5000) and anti-rabbit secondary antibody (Santa Cruz Biotechnology; 1:10000 or Merck; 1:20000). ZFP226 detection was conducted using anti-HA antibody (Cell Signaling; 1:1000) and anti-mouse secondary antibody (Santa Cruz Biotechnology; 1:20000). Western blots were repeated at least three times and band intensities were quantified using ImageJ^{51 (link)}.

Rabbit polyclonal MST2 phospho-T336 antibody was previously described (Bae et al., 2017 (link)). Rabbit polyclonal SAV1 phospho-T26 was raised against the SAV1 phospho-peptide with the sequence of VKKEpTSPLLC at an on-campus facility. The following antibodies were purchased from the indicated sources: anti-pMST1/2 (T183/180; GTX133948, GeneTex); anti-STRIP1 (A304-644A) and anti-CCM3 (A304-798A, Bethyl Laboratories Inc); anti-Tubulin (ab4074), anti-SIKE1 (ab121860), anti-STK25 (ab157188) and anti-SLMAP (ab56328, Abcam); anti-MYC (Roche); anti-FLAG (F1804, Sigma); anti-HA (sc-805), anti-PP2AA (sc-6112), anti-SAV1 (sc-101205), anti-STRN3 (sc-13562) and anti-YAP (sc-101199, Santa Cruz Biotechnology); anti-MOB4 (A4590, ABclonal); anti-GM130 (12480), anti-MST1 (3682), anti-MST2 (3952), anti-MST3 (3723), anti-MST4 (3822), anti-GAPDH (2118), anti-MOB1 (13730), anti-pMOB1 (T35;8699), anti-LATS1 (9153), anti-pLATS1/2 (HM; 8654, AL;9157), anti-pYAP (4911), anti-PP2AC (2259), anti-rabbit immunoglobulin G (IgG) (H+L) (Dylight 800 or 680 conjugates), and anti-mouse IgG (H+L) (Dylight 800 or 680 conjugates, Cell Signaling). Alexa Fluor 647 Phalloidin (A22287) was purchased from Thermo Scientific. Phos-tag conjugated acrylamide was purchased from Wako chemicals. Latrunculin B (ab144291) and cytochalasin D (sc-201442) were purchased from Abcam and Santa Cruz Biotechnology, respectively.

HFF cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Sigma) containing a protease and phosphatase inhibitor cocktail (Pierce Biotechnology, Waltham, MA). Equal amounts of protein (20 to 40 μg, depending on the target protein) were resolved in 8 to 10% (wt/vol) SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (PALL, Cortland, NY). The membranes were blocked with TBST (150 mM NaCl, 10 mM Tris-HCl, 0.1% [vol/vol] Tween 20, pH 8.0) containing 5% (wt/vol) skim milk and analyzed with the following primary antibodies: anti-YAP (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, sc-101199), anti-STING (1:500, R&D systems, Minneapolis, MN, MAB7169), anti-IE1 (1:1000, Millipore, Billerica, MA, MAB8131), anti-pp52 (1:1000, Virusys, Sykesville, MD, ICP36), anti-pp28 (1:1000, Santa Cruz Biotechnology, sc-69749), and anti-β-actin (1,10,000, Santa Cruz Biotechnology, sc-47778). All blots were incubated overnight with primary antibody at 4°C with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies for 2 h at room temperature. The enhanced chemiluminescence system (Atto, Tokyo, Japan) and X-Omat film (Kodak, Rochester, NY) were used to visualize the protein bands.

IF and IHC were performed according to the conventional protocols. The primary antibodies used for IF were anti-YAP (Abcam, Hong Kong, China #ab52771) and anti-βTrCP (Abcam, #ab233638). The primary antibodies used for IHC were: anti-YAP (Santa Cruz Biotechnology, Santa Cruz, CA, USA, #sc-101199) and anti-ISG15 (Abcam, #ab233071). For IB, the proteins were resolved on SDS-PAGE gels according to the conventional protocols. The primary antibodies used were anti-ISG15 (Abcam, #ab233071), anti-YAP (Abcam, #ab52771 and Santa Cruz, #sc-101199), anti-GAPDH (CST, #5174 and #51332), anti-Ub (Abcam, #ab7780 and #ab7254), anti-PSMB5 (Abcam, #ab167341), anti-βTrCP (Abcam, #ab71753 and #ab233638), anti-YAP^O241 (developed by Biolynx, Hangzhou, China), anti-YAP^P127 (Abcam, #ab76252), anti-YAP^P397 (CST, Boston, MA, USA, #13619), anti-HA (Abcam, #ab9110 and #ab18181), anti-TEAD4 (Abcam, #ab197589 and #ab58310), anti-6PGL (Abcam, #ab229872), anti-FLAG (CST, #8146 and #2368), anti-UbCH8 (Abcam, #ab177485), anti-HERC5 (Invitrogen, Carbsland, CA, USA, #703675), anti-ATG5 (Abcam, #ab221604), anti-LATS1 (Abcam, #ab243656), anti-CK1 (Abcam, #ab270997 and #ab115293), anti-SMAD2 (Abcam, #ab40855), anti-alpha fetoprotein (AFP, Abcam, #ab284388) and anti-albumin (Alb, Abcam, #ab207327). ELISA kits (Yingxin, Shanghai, China) were used to measure the concentration of YAP protein and Rib-5-P.

Standard western-blot assays were used to analyze protein expression in cells. The following antibodies were used for assays: anti-Flag-M2 (A8592, Sigma, 1:1000), anti-HA (2013819001, Roche, 1:1000) anti-Myc (9E10, Santa Cruz, 1:1000), anti-GAPDH (0411, Santa Cruz, 1:1000), anti-PARK2 (Abcam, ab15954), anti-YAP (63.7, Santa Cruz, 1:1000), Protein signals were detected with an ECL kit ( Millipore Co., Billerica, Massachusetts, USA).