Mouse anti desmin

The cells grown on coverslips were fixed in 4% (v/v) paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature or overnight at 4°C. After rinsing in PBS, the fixed cells were permeabilized and nonspecific epitopes were blocked using 2% bovine serum albumin (Bovogen Biologicals, Keilor East, Vic, Australia) in 0.1% Tween-20/PBS, followed by incubation in the diluted primary antibody for 1 h at room temperature or overnight at 4°C. After three washes in PBS, samples were incubated for 1 h at room temperature with secondary antibodies diluted in PBS. Prepared samples were then mounted using Vectashield medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA) and photographed using a fluorescence microscope (Nikon Corporation, Tokyo, Japan). The manufacturers and catalog numbers of the antibodies employed were as follows: mouse anti-myosin heavy chain (MHC; cat. no. MAB4470; R&D Systems) mouse anti-myogenin (cat. no. ab-1835; Abcam, Cambridge, MA, USA), mouse anti-desmin (cat. no. D1033; Sigma-Aldrich, St. Louis, MO, USA), Alexa-568 goat anti-mouse IgG (cat. no. A-11004), Alexa-488 goat anti-rabbit IgG (cat. no. A-11008) (both from Life Technologies). Quantification of immunofluorescence staining confirmed that four slides were used for each condition. Graphs represent the average of multiple tests from three independent experiments.

The following primary antibodies were used: NW6 Rabbit anti-DP was generated using anti-human DP CT fragment found in both human DPI and II (Angst et al., 1990 (link)), anti–phospho-S2849 DP antibody was generated using rabbits raised against a DP peptide phosphorylated at S2849 (Bouameur et al., 2013 (link)), mouse anti-S-Tag (EMD Millipore), M2 mouse anti-FLAG (Abcam), rabbit anti-FLAG (Abcam), DM1A mouse anti–α-tubulin (Abcam), HECD-1 mouse anti–E-cadherin (Abcam), rabbit anti-PRMT-1 (Abcam), mouse anti-desmin (Abcam), rabbit anti-GAPDH (Sigma-Aldrich), rabbit anti-HA (Sigma-Aldrich), KSB17.2 mouse anti-K8/K18 (Sigma-Aldrich), C2206 rabbit anti–β-catenin (Sigma-Aldrich), mouse anti-tubulin (University of Iowa Developmental Studies Hybridoma Bank), mouse anti-GSK3 and rabbit anti-pGSK3 (Cell Signaling Technology), JL-8 mouse anti-EGFP (Takara Bio Inc.), and 110B7E rabbit anti-phospho Substrate (RXXS*/T*; Cell Signaling Technology). The following secondary antibodies were used: Alexa Fluor 568 goat anti–mouse IgG at 1:300 (Life Technologies) and HRP-conjugated goat anti–mouse and goat anti–rabbit IgG at 1:5,000 (KLP Labs). Dispase Enzyme was purchased from Roche.