Rabbit anti psmad1 5 9

Cells were lysed with RIPA buffer (Sigma) containing protein inhibitors (complete ULTRA Tablets, Roche, Basel, Switzerland) and phosphatase inhibitors (PhosSTOP, Roche, Basel, Switzerland). Samples were denatured by incubating at 95 °C for 5 min in sample buffer and separated by using SDS-PAGE (precast 8–16% gradient gels, Biorad, Hercules, CA, USA). Then, the samples were transferred to a PDVF membrane (Millipore). The membrane was blocked with Odyssey Blocking solution (Li-Cor Biosciences, Lincoln, NE, USA) for 1 h at room temperature, followed by primary antibody incubation at 4 °C overnight. The primary antibodies used in the present study included rabbit anti-p-Smad1/5/9 (Cell Signaling Tech, Danvers, MA, USA), rabbit anti-Smad 1(Cell Signaling Tech), mouse anti-beta actin (Cell Signaling Tech), rabbit anti-p-Smad 2 (Cell Signaling Tech) and rabbit anti-Smad 2 (Cell Signaling Tech). The proteins were detected by Odyssey system (Li-Cor bioscience) followed by the secondary antibodies including IRDye 680-conjugated goat anti-rabbit IgG (Li-Cor Bioscience) and IRDye 800CWconjugated goat anti-mouse IgG (Li-Cor Bioscience, Lincoln, NE, USA).

For immunofluorescence, all steps were performed within the microfluidic device at room temperature, unless otherwise stated. Cells were permeabilized with 0.1% (v/v) Triton X-100 (Cat: T8787, Sigma-Aldrich) in PBS (PBST) for 15 min and incubated with 10% (v/v) goat serum (Cat: G9023, Sigma Aldrich) in PBST for 30 min. Cells were incubated overnight at 4°C with rabbit anti-pSMAD1/5/9 (1:600 dilution, Cat: 13820, Cell Signaling Technology, Danvers, MA, USA). The secondary antibodies goat anti-rabbit IgG-FITC (Cat: 111-095-045) or goat anti-rabbit IgG-Rhodamine Red™-X (Cat: 111-295-045) (Jackson ImmunoResearch Laboratories, Westgrove, PA, USA) were diluted 1:500 in PBST and applied for 1 h. Cells were then washed with PBST and simultaneously stained for 10 min for nuclei (DAPI) and, when needed, actin filaments (Acti-stain 670 phalloidin, 1:600 dilution; Cat: SKU PHDN1, Cytoskeleton, Denver, CO, USA). Imaging was performed using a spinning disk confocal microscope (Yokogawa CSU-X1, Hamamatsu X2 EMCCD mounted on a Leica DMI-6000).

Near-confluent monolayer cultures of FACS-isolated FAPs in 6-well plates were serum starved for 2 hours and then incubated for 1 hour with or without ligands and antibodies at the indicated concentrations. In the JAB0505 plus ActA-mAb condition, FAPs were incubated with ActA-mAb 10 minutes prior to, as well as during, the 1-hour incubation with JAB0505 to allow time to sequester endogenous activin A. Procedures for cell lysis, SDS-PAGE, electrophoretic transfer, antibody incubation, and washing were previously described (6 (link)). Total protein concentration was measured using the DC protein assay (Bio-Rad), and equal amounts of total protein were loaded per lane of the same gel. Antibodies used include rabbit anti–p-SMAD1/5/9 (8 (link)) (Cell Signaling Technology [CST], 13820), mouse anti–β-actin (CST, 3700), rabbit anti–mouse IgG (H+L), Alexa Fluor 647 (Thermo Fisher Scientific, A-21239) (secondary antibody for β-actin detection), and anti–rabbit IgG, HRP-linked (CST, 7074) (secondary and tertiary antibody for p-SMAD1/5/8 and β-actin detection, respectively). After the final wash, membranes were incubated in in SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) and imaged using the IVIS SpectrumCT (PerkinElmer). p-SMAD1/5/8 and β-actin bands were imaged separately, as separate secondary antibody incubations of the same membrane were required.