Anti myc

WB and IFA were performed as described with antibodies, and the dilutions used were as follows: anti-Myc (Cell Signaling) 1:3000; anti-HA (Cell Signaling) 1:3000, anti-H3 (Abcam) 1:10000, anti-BIP 1:1000 (polyclonal), anti-H2A. Z (polyclonal–kindly provided by Dr. Nicolai Siegel), anti-His Tag 1:3000 (Cell Signaling); and anti-GAPDH 1:4000 (polyclonal) [89 (link)]. For IFA of myc-tagged parasites, after fixation with 4% paraformaldehyde and permeabilization with Triton X-100, an overnight incubation with anti-Myc (Cell Signaling) antibody 1:3000 in 1% PBS/BSA at 4°C followed by secondary antibody conjugated to Alexa Fluor 555 (Thermo Fisher) at 1:500 in PBS/BSA 1% was performed. When mentioned, IFA using anti-GP90 and anti-GP82 1:1000 (kindly provided by Nobuko Yoshida) was performed as described previously [90 (link)]. Slides were further mounted with Vectashield plus DAPI (Vector laboratories) and visualized at 100 X magnification in an inverted OLYMPUS microscope model IX81 with Z axis motorization. Images were acquired using OLYMPUS CELL R version software 3.2.

Whole cell lysates for Western analysis and immunoprecipitations were prepared in radioimmunoprecipitation assay buffer (RIPA) and isotonic immunoprecipitation buffer, respectively [28] (link). Immunoprecipitations were performed as previously described [29] (link). Briefly, whole cell lysates were incubated overnight with anti-HA (HA.11, Covance) or anti-Myc (Cell Signaling, 2278) antibodies, and the immune complexes were precipitated using protein A/G-Sepharose, followed by SDS-PAGE and Western blotting analysis using the following primary antibodies: anti-HA (Cell Signaling, 2367); anti-Myc (Cell Signaling, 2278); anti-Flag M2 (Sigma, F3165); anti-β actin (Sigma, A5441); anti-PP2A/A (Santa Cruz, sc-6112); anti-Cα (BD Transduction Laboratories, 610556). After incubation with the primary antibodies and corresponding HRP-conjugated secondary antibodies, the blots were developed by enhanced chemiluminescence.

Epitope-tagged Prdm6 and MRTF-A expression constructs were transfected into COS-7 or 10T/12 cells. After 48 hours cells were lysed in RIPA buffer plus protease and phosphatase inhibitors. Approximately 1 mg of protein was incubated with 3 to 5 μg of anti-flag (Cell Signaling Technology; catalog 8146S), anti-myc (Cell Signaling Technology; catalog 2276s), or nonimmune mouse IgG (MilliporeSigma; catalog NI03) overnight at 4°C with rotation. Immunoprecipitants were washed 3 times in wash buffer (20 mM Tris HCl, pH7.3, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100), eluted in sample buffer, resolved by SDS-PAGE, transferred to nitrocellulose, and probed with anti-flag (Cell Signaling Technology; catalog 8146S), anti-myc (Cell Signaling Technology; catalog 2276s), or anti-MRTF-A (sc-32909, Santa Cruz Biotechnology) Abs. For Western blots used to measure SMC differentiation marker gene expression, cleared RIPA lysates were boiled in sample buffer, run on an 10% SDS-PAGE gel, transferred to nitrocellulose, and probed with the following Abs: SM α-actin (MilliporeSigma, catalog A5228), CNN1 (LifeSpan Bio catalog LS-B7497), SM22 (Santa Cruz Biotechnology; catalog sc-271719), and GAPDH (Cell Signaling Technology; catalog 97166s).