Anti ha

Cells were grown in 6-well culture plates and harvested at 80% confluence to prepare whole-cell lysates using modified RIPA buffer containing Triton X-100 [24 (link)]. The spheroids cultured in the 3D collagen type I environment were harvested as described in a previous report [28 (link)]. Primary antibodies used for immunoblotting were as follows: phospho-Y⁴¹⁶ Src, (Cell Signaling, Boston, MA, USA); c-Src (Santa Cruz Biotechnology, Santa Cruz, CA, USA); FAK, phospho-Y³⁹⁷FAK (BD Bioscience, San Jose, CA, USA); anti-HA (BioLegend, San Diego, CA, USA).

Kc167 cells were collected, washed in PBS and incubated for 30 min in IP buffer (150 mM NaCl, 0.5% NP40, 50 mM Tris-HCl, pH8.0, 1mM EGTA) supplemented with protease inhibitor cocktail (Roche). The extracts were cleared by centrifugation at 13.000g for 15 min at 4°C and subjected to SDS-PAGE (50 μg of proteins par lane) or immunoprecipitation (1 mg per point). For immunoprecipitation, proteins were preadsorbed with 100 μl of sepharose beads slurry for 1h at 4°C before being incubated with 20 μl of anti-GFP (Chromotek), anti-V5 (Sigma-Aldrich) or anti-HA (Covance) antibody coupled to sepharose beads, or with 10 μl of rabbit anti-MLF [19 (link)] or rabbit IgG (SantaCruz) in the presence of 20 μl of protein A sepharose beads (Sigma), for 4h at 4°C. The beads were spun down and washed in IP buffer and immunoprecipitated proteins were processed for SDS-PAGE and Western Blot analyses. Western blots were performed using standard techniques and the blots were developed by photoluminescence procedure using Lumi-Light^PLUS Western Blotting Substrate (Roche) and Amersham Hyperfilm^TM ECL (GE Healthcare) or Chemidoc Touch Imaging System (BioRad). The following antibodies were used for Western blots: anti-V5 (Invitrogen), anti-HA (BioLegend), anti-GFP, anti-tubulin (Sigma-Aldrich), anti-Renilla luciferase (MBL), and anti-MLF [19 (link)].

Standard methods were used for the preparation and ligation of DNA fragments and for transformation and recovery of plasmid DNA from Escherichia coli [44 ]. Yeast was transformed by the LiAc procedure of Schiestl and Gietz [45 (link)]. Proteins were separated by SDS–PAGE [42 (link)]. Western blots were treated with rabbit polyclonal antibodies against anti F₁-β (a gift from Dr. Alexander Tzagoloff), anti-Atp6 (a gift from Dr. Jean-Paul di Rago and Dr. Marie-France Giraud), anti-HA (Biolegend, San Diego, CA, USA), and VDAC1 (Invitrogen, Waltham, MA, USA). The blots were then followed by a second reaction with peroxidase-coupled anti-rabbit IgG. The antibody complexes were visualized with the super signal chemiluminescent substrate kit (Pierce Chemical, Dallas, TX, USA). The protein concentrations were determined by the method of Lowry et al. [40 (link)].

Cells were lysed in RIPA buffer containing protease inhibitors for 30 min on ice, followed by centrifugation at 15000 × g for 30 min. After SDS-PAGE, extracted proteins were transferred onto PVDF membranes (GE Healthcare) and probed with primary antibodies at 4°C overnight. The membranes were then incubated with secondary antibodies for 1 h, and the bands were captured through an ECL HRP substrate (Millipore). Anti-PRMT5 (Abcam, ab109451), anti-CDK4 (Santa Cruz Biotechnology, sc-260), anti-β-actin (Cell Signaling Technology, #4967), anti-β-tubulin (Cell Signaling Technology, #2146), anti-Flag M2 (Sigma-Aldrich, F3165), anti-HA (Biolegend, 901503), anti-6× His (Proteintech, 66005-1-Ig), anti-pRB S780 (Abcam, ab47763), anti-Mono-Methyl Arginine (Cell Signaling Technology, #8015) and anti-SDMA (Millipore, 07-413), anti-CCNE1 (Proteintech, 11554-1-AP), anti-CDC6 (Proteintech, 11640-1-AP) antibodies were purchased.

Cells were grown in six‐well plates to 80% confluency and harvested. Cell pellet was snap frozen and lysed in 100 μl lysis buffer (0.5% NP40, 50 mM HEPES (pH 7.5), 150 mM NaCl, 50 mM NaF, 400 nM Na₃VO₄, 1 mM PMSF and protease inhibitor cocktail). The cell lysate was cleared by centrifugation (15,000 g for 20 min), boiled for 5 min after the addition of 3× Laemmli sample buffer, loaded on NuPAGE 4–12% Bis‐Tris SDS–PAGE gels (Invitrogen) for gel electrophoresis and then transferred onto nitrocellulose membranes (Trans‐Blot Turbo, BioRad). The following primary antibodies were used: anti‐PTPN14 (#13808, Cell Signaling), anti‐actin (#179467, Abcam), anti‐YAP1 (#15407, Santa Cruz), anti‐YAP1phosphoS127 (#4911, Cell Signaling), and anti‐HA (HA.11,901513, BioLegend). Proteins were detected by enhanced chemiluminescence (ECL, Amersham) using horseradish‐peroxidase‐coupled secondary antibodies (Rabbit #7074, Cell Signaling and Mouse #115035003, Jackson ImmunoResearch).