Sc 805

Immunoblotting was performed as previously described [26 (link)]. Briefly, inputs or immunoprecipitates were separated by SDS-PAGE and blotted using antibodies to spinophilin, NF-M, V5-tag, HA-tag (as above or rabbit HA (SC-805, Santa Cruz Biotechnology)), PP1γ1, pan PP1 (SC-7482, Santa Cruz Biotechnology), PKA substrate antibody (#9624, Cell Signaling), Myc (sc-40, Santa Cruz Biotechnology), or tyrosine hydroxylase (TH) antibody (#22941, ImmunoStar, Hudson, WI). Appropriate infrared secondary antibodies were used (donkey anti-goat, donkey anti-rabbit, or donkey anti-mouse conjugated to Alexa Fluor 690 or 780; ThermoFisher or Jackson Immunologicals), and fluorescence intensity measurements were made using Image Studio (LI-COR Biosciences, Lincoln, NE).

Cells were grown on coverslips, fixed with 4% paraformaldehyde and permeabilized with ice-cold methanol. Cells were rinsed with phosphate-buffered saline, blocked with 10% goat serum and then incubated with primary antibody overnight, followed by incubation with Alexa Fluor-conjugated secondary antibodies (Life Technologies). Coverslips were mounted with ProLong Gold Antifade reagent with DAPI (Life Technologies). The following primary antibodies were used for immunofluorescence: anti-Flag (Sigma-Aldrich, F1804, 1:400), anti-HA (Santa Cruz Biotechnology, sc-805, 1:400), anti-PML (Santa Cruz Biotechnology, sc-966, 1:400) and anti-PP1α (Bethyl Laboratories, A300-904A, 1:400). The stained slides were visualized by a bright-field or confocal microscope. Senescence-associated β-galactosidase activity in prostate tissue was measured with the senescence detection kit (Calbiochem) on 5 μm thickness frozen section.

Frozen TA muscles were cut transversally, fixed in 4% PFA for 20 min and permeabilized with methanol for 6 min at −20 °C. Muscle sections were then blocked with a solution containing 4% BSA in PBS followed by incubation with anti-mouse AffiniPure Fab fragment (Jackson) to avoid unspecific binding. Immunostaining with primary antibodies was performed overnight at 4 °C. Secondary antibodies coupled to Alexa Fluor 488 or 594 (Molecular Probes) were used to reveal antibody binding. Nuclei were visualized by counter staining with DAPI. Cultured cells were fixed in 4% PFA, permeabilized with either 0.25% Triton (for phopho-p38 antibody) or methanol and blocked with 4% BSA in PBS before antibody incubation. Immunostaining and detection was performed as above. Primary antibodies used were: anti-laminin (Sigma, L9393; dilution 1:1,000), anti-PJA1 (Proteintech, 17687-1-AP; dilution 1:50), anti-MYOD (BD, 554130; dilution 1:50), anti-EZH2 (AC22, Cell Signaling; dilution 1:150), anti-MyHC (MF-20, DSHB; dilution 1:30 and Santa Cruz, SC-20641; dilution 1:100), anti-KI67 (BD 556003; dilution 1:1.000), anti-HA (Santa Cruz, SC-805 and SC-7392; dilution 1:50) and anti-phospho-p38 (Cell Signalling, D3F9; dilution 1:150). Images were acquired with a Leica confocal microscope and edited using the Photoshop CS4 software. Fields reported are representative of all examined fields.

Embryos were co-injected at one-cell stage with Flag-smad1, HA-foxd4l1.1 and Myc-xbra mRNA constructs in four different groups. The injected embryos were collected at stage 11.5. They were then homogenized in lysis IP buffer. The composition of the IP buffer is described in Kumar et al. (2018)^{15 (link)}. Cell lysates were cleared by centrifugation and were then incubated with Flag-Smad1 (F-2574, Sigma) monoclonal antibody and α-HA (SC-805, Santa Cruz Biotechnology) polyclonal antibody overnight at 4 °C with the immunocomplexes precipitated by protein A/G beads (SC-2003, Santa Cruz Biotechnology). Proper amounts of precipitated beads-protein complex were boiled in the sample buffer, and resolved by electrophoresis in 10% SDS–polyacrylamide gels. Western blotting of Myc-Xbra (SC-789, Santa Cruz Biotechnology) was performed by using an anti-Myc and secondary antibody anti-mouse (SAB-100, Stressgen, Victoria, BC). Immune complexes were visualized by using an ECL detection kit (GE Healthcare).