Journal: Cell reports

Article Title: Single-cell analysis of the human pancreas in type 2 diabetes using multi-spectral imaging mass cytometry

doi: 10.1016/j.celrep.2021.109919

Figure Lengend Snippet:

Article Snippet: 154Sm-Beta-actin (Clone 2F1–1) , Fluidigm , Cat#3154021D.

Techniques: Software

Journal: Molecular Biology of the Cell

Article Title: ECM stiffness primes the TGFβ pathway to promote chondrocyte differentiation

doi: 10.1091/mbc.E12-03-0172

Figure Lengend Snippet: Smad3 phosphorylation, localization, and transcriptional activity are sensitive to ECM stiffness. (A) Western analysis of whole-cell lysates from ATDC5 cells cultured as indicated for 24 h reveals that C-terminal Smad3 phosphorylation (top) and total protein (middle) are increased specifically on a 0.5-MPa substrate, whereas β-actin levels (bottom) remain unchanged. (B) Immunofluorescence microscopy allows visualization of stiffness and TGFβ-sensitive Smad2/3 localization (green) relative to DAPI-stained nuclei (red). (C) Relative to cells grown on plastic, a greater percentage of primary chondrocytes and ATDC5 cells have nuclear Smad2/3 when grown on 0.5-MPa substrates, particularly in the absence added TGFβ. (D) In transiently transfected ATDC5 cells grown for 48 h on 0.5-MPa substrates, the activity of the TGFβ-responsive SBE-luciferase promoter reporter construct is increased relative to cells grown on plastic. Luciferase activity is normalized to expression of β-galactosidase from a cotransfected control construct. * p < 0.05, † p < 0.01.

Article Snippet: Blots were probed with the following primary and secondary antibodies: β-actin (ab8226; Abcam, Cambridge, MA), Smad2/3 (sc8332; Santa Cruz Biotechnology, Santa Cruz, CA), pSmad3 (a gift from E. Leof, Mayo Clinic, Rochester, MN), p38 (9218; Cell Signaling, Beverly, MA), pp38 (9211; Cell Signaling), and anti-mouse and anti-rabbit secondary antibodies that were conjugated to 680 or 800CW IRDye fluorophores detected using a LI-COR infrared imaging system (926-68020 and 926-32211; LI-COR Biosciences, Lincoln, NE).

Techniques: Activity Assay, Western Blot, Cell Culture, Immunofluorescence, Microscopy, Staining, Transfection, Luciferase, Construct, Expressing