Anti α tubulin clone b512

CAFs were harvested in Laemmli lysis buffer (0.125 M Tris-HCl, 10% glycerol, 2.3% sodium dodecyl sulfate (SDS), pH 6.8). Lysates were suspended in reducing sample buffer (0.5 M Tris-HCl, 40% glycerol, 9.2% SDS, 4.5% 2-mercaptoethanol, 0.011% bromophenolblue, pH 6.8) and boiled at 95°C for 5 minutes. Equal protein amounts were separated on 8% polyacrylamide gels, transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), blocked in 5% nonfat milk in phosphate-buffered saline (PBS) with 0.5% Tween-20 (Sigma-Aldrich), and immunostained. The following antibodies were used: anti-α Smooth Muscle Actin (SMA, clone 1A4, Sigma-Aldrich), anti-biglycan (H-150, SantaCruzBiotechnology, Santa Cruz, CA, USA), anti-decorin (clone 115402, R&D Systems), anti-versican (H-56, SantaCruz Biotechnology). Detection was performed by horseradish peroxidase-conjugated secondary antibodies (all GE Healthcare, Pittsburgh, PA, USA) and chemiluminescence (Pierce ECL Western blotting substrate, Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions. Membranes were stripped with ReBlot Plus mild antibody stripping solution (Merck Millipore) and reprobed with anti-α-tubulin (clone B-5-1-2, Sigma-Aldrich) as a loading control. Quantification of protein expression was performed with ImageJ software (NIH, MD, USA).

The following antibodies were used. Anti-SERT rabbit polyclonal antibody/AB10514P (EMD Millipore, Billerica, MA); anti-P-gp mouse monoclonal antibody Clone 2F7 (OriGene, Rockville, MD); anti-MRP3 rabbit anti-human antibody LS-C177398 (Lifespan Biosciences Inc, Seattle, WA); anti-NET rabbit polyclonal antibody LS-C101935 (Lifespan Biosciences Inc, Seattle, WA) and anti-BCRP mouse monoclonal antibody BXP-21 (Abcam, Cambridge, MA). Loading control antibodies included: Anti-GAPDH (6C5, sc-32233, Santa Cruz Biotechnologies, Santa Cruz, CA); mouse monoclonal Grb2 (BD Biosciences, San Jose, CA), and anti-α-Tubulin clone B512 (Sigma-Aldrich Co, St. Louis, MO). Primary antibodies were diluted according to the manufacture’s suggestion (1:1,000). IRDye® 800CW Goat Anti-Rabbit and IRDye® 680RD Goat Anti-Mouse Li-COR dyes (LI-COR, Inc., Lincoln, NE) were diluted at 1:20,000. RFU readings were normalized to control housekeeping proteins (Grb2, Tubulin, or GAPDH) in qWestern-blot assays.

Cell pellets were lysed in Laemmli buffer ×2 (Tris–HCl 0.5 M, pH 6.8; 0.5 M DTE; 0.5% SDS) and protein concentrations determined using the Bradford reagent (Bio-Rad). Protein extracts were separated on SDS–PAGE (8% or 15% or 4–15% gradient [vol/vol]) gels. Gels were transferred onto nitrocellulose membranes (GE HealthCare and Bio-Rad) for immunoblotting with the following antibodies: anti-NLRP3 (Cryo-2, 1:1,000) and anti-caspase-1 (Bally-1, 1:1,000) from AdipoGen, anti-ASC (1:2,000) from Enzo Life Science, and anti-γH2AX (JBW301, 1:1,000), anti-P-Ser15-p53 (1:1,000), and anti-ATM Ser1981 (10H11.E12, 1:1,000) from Millipore. Anti-P-KAP1 Ser824 (1:1,000), anti-KAP1 (1:1,000), and anti-NEK7 (A302-684A, 1:1,000) from Bethyl Laboratories, anti-p53 (clone DO7 1:2,000) and anti-NOXA (114C307, 1:1,000) from Santa Cruz; anti-ATM (#ab32420, 1:1,000), anti-fibrillarin (ab4566, 1:1,000), and anti-GAPDH (ab9484, 1:1,000) from Abcam; anti-FLAG (F7425 1:5,000) and anti-α-tubulin (clone B-5-1-2 1:1,000) from Sigma-Aldrich; anti-XPO2 (GTX102005 1:1,000) and anti-IPO5 (GTX114515 1:1,000) from Genetex, and anti-actin (C4, 1:100,000) from MP Biomedical. The Fiji and Image Laboratory (Bio-Rad) programs were used for densitometric analysis of immunoblots, and the quantified results were normalized as indicated in the figure legends.

Glass coverslips (Carl Zeiss) were cleaned in 1M KOH and sonicated for 15 min, coated with poly-L-lysine for 15 min, washed with PBS, coated with 10 μg/ml of either anti-murine CD3ε (clone 145-2C11) or CD25 (clone 3C7) (Becton Dickinson) overnight at 4°C, rinsed in PBS, and blocked with RPMI 10% FCS. 6–8 days after stimulation, OT-I CTLs were added dropwise and allowed to adhere for 3 min. Cells were extracted in 50 mM PIPES, 2.5 mM MgCl₂, 2.5 mM EGTA, 0.25% glutaraldehyde, and 0.5% Triton X-100 for 1 min immediately prior to fixation in 4% paraformaldehyde for 15 min. They were then stained with phalloidin-Alexa 488 (Invitrogen), anti-α-tubulin (clone B-5-1-2, Sigma), and Alexa-568-labeled anti-mouse immunoglobulin G (IgG). Images were obtained on a Zeiss Elyra structured illumination microscope and processed with Imaris software (Bitplane).