Rabbit anti vinculin

The cells were lysed after the 24 h treatment and the proteins were separated using electrophoresis, as previously described [38 (link)]. The primary antibodies were: rabbit anti-p21 (1:1000, Cell Signaling Technology, distributed by Euroclone, Milan, Italy), rabbit anti-phospho Rb (1:1000, Cell Signaling Technology) and rabbit anti Rb (1:1000, Cell Signaling Technology). The membrane was washed in a T-PBS buffer, then incubated for 1 h at RT with a goat anti-rabbit IgG Alexa Fluor 750 antibody or with a goat anti-mouse IgG Alexa Fluor 680 antibody (Invitrogen, Milan, Italy) and then visualized using an Odyssey Infrared Imaging System (LI-COR^® Bioscience, distributed by Carlo Erba, Milan, Italy). The rabbit anti-vinculin (1:1000, Cell Signaling Technology) was used in order to assess the equal amounts of protein loaded in each lane.

Huh7 cells and FLC PDX spheroids were lysed in lysis buffer (RIPA buffer (Sigma, St. Louis, MO), 25x Complete Protease Inhibitor Cocktail (Sigma), 100x Pierce Phosphatase Inhibitor (Thermo Fisher Scientific), 100 mM phenylmethanesulfonyl fluoride, β-mercaptoethanol, 1 M dithiothreitol) at 4 °C. All lysates were flash frozen, thawed, and centrifuged for 10 minutes at 14,000 × g at 4 °C. Protein concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were diluted 1:1 in Laemmli Sample Buffer (Bio-Rad) containing 5% β-mercaptoethanol, heated at 95 °C for 3 minutes, loaded into 12% Mini-PROTEAN TGX Precast Gel (Bio-Rad), and run in 1X Tris/Glycine/SDS buffer (Bio-Rad) for 75 minutes at 150 V. Transfer was performed with the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with either 4% milk or bovine serum albumin (BSA), probed with primary mouse anti-CA12 (1:500, ab140385, Abcam, Cambridge, MA) or rabbit anti-vinculin (1:1000, #4650, Cell Signaling Technology, Danvers, MA), and then incubated with horseradish peroxidase conjugated secondary antibodies. Membranes were incubated in ECL Prime Western Blotting Detection System (GE Healthcare, Little Chalfont, UK) for 5 minutes before visualization.

Cells were treated with 4NQO ± VE-821 at the indicated doses for 1 h before cell lysates were harvested. Primary antibodies: goat anti-ATR (1:500, # 1887, Santa Cruz, Dallas, TX, USA), rabbit anti-CHK1 (Ser345) (1:1000, # 2341, Cell Signalling, Cell Signalling, Danvers, MA, USA) and rabbit anti-vinculin (1:1000, # 4650, Cell Signalling).

HEK293T cells were collected at 36 hours after transfection, and cells were lysed in radio-immunoprecipitation assay lysis buffer containing protease inhibitor cocktail (B14001, Bimake). Equal amounts of samples were electrophoresed on SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in PBS with 0.1% Tween 20 and then incubated with primary antibodies diluted in 5% BSA at 4°C overnight. The membranes were visualized using ECL Western Blotting Substrate (180-501, Tanon) after incubation with HRP-conjugated secondary antibody for 2 hours at room temperature. Digital images were obtained from a Tanon 5200s Imaging Workstation. The following primary antibodies were used for Western blots: rabbit antibody against KPNA7 (HPA031395, Sigma-Aldrich) at 1:1000 dilution, rabbit anti-FLAG (F7425, Sigma-Aldrich) at 1:3000 dilution, rabbit anti-HA (3724, Cell Signaling) at 1:2000 dilution, rabbit anti-vinculin (13901, Cell Signaling) at 1:3000 dilution, mouse anti-GST (A00865, GenScript) at 1:1000 solution, and rabbit anti-KPNA2 (ab70160, Abcam) at 1:1000 dilution.

Lgals3‐R200S wild‐type, heterozygous and mutant MEFs were harvested from 13‐day mouse embryos. Cells were cultured on 10 cm dishes until they reached 70% confluency. Experiments were carried out at 0–4 °C to reduce biotin internalization. Cells were washed three times with ice‐cold PBS pH 8.0 and labeled with 1 mg·mL⁻¹ EZ‐Link™ Sulfo‐NHS‐LC‐Biotin (Thermo Fisher Scientific) for 30 min with gentle rocking. The cells were then washed three times with ice‐cold PBS containing 100 mm glycine, and lysed in 50 mm sodium phosphate dibasic, 1 mm sodium pyrophosphate, 20 mm sodium fluoride, 2 mm EDTA, 2 mm EGTA, 1% Triton X‐100, 0.5 mm DTT, and complete protease inhibitor cocktail (Roche, Basel, Switzerland) lysis buffer. A portion of the whole cell lysate was kept as an input control. Remaining lysate was incubated with NeutrAvidin agarose beads (Thermo Fisher Scientific) for 3 h with slow rotation prior to washing three times with lysis buffer. Samples were boiled in loading buffer containing DTT prior to SDS/PAGE and western blot. Antibodies used were as follows: goat anti‐Galectin 3 (AF1197, R&D Systems, Minneapolis, MN, USA) diluted 1 : 10 000 and rabbit anti‐Vinculin (#4650, Cell Signaling Technology, Danvers, MA, USA) diluted 1 : 1000.