mouse monoclonal antibody against rab5a  (Proteintech)

Journal: Bioactive materials

Article Title: Matrix stiffness exacerbates the proinflammatory responses of vascular smooth muscle cell through the DDR1-DNMT1 mechanotransduction axis.

doi: 10.1016/j.bioactmat.2022.01.012

Figure Lengend Snippet: Fig. 1. Substrate stiffness-induced DDR1 activation is independent of collagen binding. (A) to (C) Western blot assay to determine phosphorylation of DDR1 at tyrosine 792 (p-DDR1) and the total DDR1 expression. GAPDH served as an internal control. Shown are representative blots from at least 3 independent experiments. Semi-quantification (in the lower panels) of indicated protein was performed by using the ImageJ software based on the analysis of the gray band intensity. (A) The p-DDR1 and total DDR1 levels in SMCs cultured for 24 h on collagen type I (Col) coated-polyacrylamide (PA) gels with a stiffness of 2 (soft) or 20 (stiff) kPa. (B) The p-DDR1 levels in SMCs grown on Col-coated gels for 24 h in the presence of the DDR1: Fc peptides. (C) The p-DDR1 and total DDR1 levels in SMCs cultured for 24 h on fibronectin (Fn)-coated PA gels. (D) Representative stimulated emission depletion microscopy (STED) images of DDR1 and Rab5A immunofluorescence in SMCs on Fn-coated gels. Cells were either pretreated with DDR1-IN-1 or DMSO. (E) Schematic diagram illustrating measuring the interaction between the collagen and the surfaces of SMCs by atomic force microscopy (AFM). (F) AFM to determine the adhesion probability and rupture forces between the Col- or BSA-coated probe and the cell surface. Each dot represents one cell. (G) and (H) AFM to determine the adhesion probability and rupture forces between the DDR1 ligand (Col) and the cell surface. In (G), each dot represents an independent experiment, and in (H), each dot represents the rupture force of a single bond between Col and the cell surface. (I) AFM to determine the adhesion probability in cells that were pretreated with DDR1-IN-1 or DMSO and cultured on Fn-coated gels. Each dot represents an inde pendent experiment. (J) Western blot assay to determine DDR1 and GAPDH in cell lysates prepared using a non-reducing condition. (K) Quantitative RT-PCR to determine the expressions of DDR1 targeted genes in SMCs on Fn-coated gels. Each dot represents an independent experiment. *P < 0.05 vs. the indicated group.

Article Snippet: Rabbit polyclonal antibodies against DNMT1, p53, and DDR1, and mouse monoclonal antibody against Rab5A were purchased from Proteintech.

Techniques: Activation Assay, Binding Assay, Western Blot, Phospho-proteomics, Expressing, Control, Software, Cell Culture, Microscopy, Immunofluorescence, Quantitative RT-PCR