Anti ha polyclonal

HeLa cells were cultured onto a cover glass in a 24-well plate for 1 day, and were transfected with plasmid for the expression of zyxin. The amount of DNA was kept constant by adding empty vector. After 24 h, MitoTracker Red (Thermo Fisher) was treated for 30 min according to manufacturer’s protocols. Cells were fixed with 4% paraformaldehyde in PBS for 30 min in room temperature, and were then permeabilized and blocked with PBS containing 0.2% of Triton X-100 and 5% FBS for 60 min. Permeabilized cells were labeled with anti-HA polyclonal (Sigma Aldrich) Ab in 1% BSA/PBS over night at 4 °C. Cells were washed with PBS and incubated with Alexa Fluor 488-conjugated Ab (Thermo Fisher) in 1% BSA/PBS for 60 min at room temperature. Stained cells were covered with Prolong Diamond Antifade Mountant with DAPI (Thermo Fisher) and observed under a FluoView FV1200 confocal microscope (Olympus).

HEK 293T whole cell extracts for co-IP were prepared using lysis buffer containing 1%TX-100, 50 mM HEPES (pH 7.4), 138 mM KCl, 4 mM NaCl, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 1 mM EGTA pH 8, 50 μM PMSF, 1 mM β-Glycerophosphate, 1 mM sodium orthovanadate, and protease inhibitors (Thermo Scientific cat # 88265) as previously described (Lamia et al., 2004). Immunoprecipitation was performed using anti-Flag M2 agarose beads (Sigma cat #A2220). Antibodies for Western Blots were anti-FLAG polyclonal (Sigma cat #F7425), anti-V5 polyclonal (Bethyl Labs cat# A190–120A), anti-HA polyclonal (Sigma cat # H6908), anti-α-TUBULIN (Sigma cat # T5168), anti-E2F1 (Santa Cruz KH95), anti-E2F8 (Abcam ab109596), CRY1-CT and CRY2-CT^{11 (link)}. For CHX assay, HEK293T cells were lysed with a RIPA Buffer containing 1% TX-100, 147 mM NaCl, 12 mM Sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 10 mM EDTA, 50 μM PMSF, 1 mM β-Glycerophosphate, 1 mM Sodium Orthovanadate and protease inhibitors (Thermo Scientific cat # 88265).

293 T whole cell extracts were prepared using lysis buffer containing 1%TX-100, 50 mM HEPES (pH 7.4), 138 mM KCl, 4 mM NaCl, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 1 mM EGTA pH 8, 50 μM PMSF, 1 mM β-Glycerophosphate, 1 mM sodium orthovanadate, and protease inhibitors (Roche cat # 11697498001 or Thermo Scientific cat # 88265) as previously described (Lamia et al.^{40 (link)}). ASF cell extracts were prepared from RIPA buffer containing 1% TX-100, 147 mM NaCl, 12 mM sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 10 mM EDTA, 50 μM PMSF, 1 mM β-Glycerophosphate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and protease inhibitors (Thermo Scientific cat # 88265). Antibodies used for immunoprecipitation were anti-FLAG M2 agarose beads (Sigma cat # A2220) and monoclonal anti-HA agarose beads (Sigma cat # A2095). Antibodies for Western blot were anti-FLAG polyclonal (Sigma cat # F7425), anti-HA polyclonal (Sigma cat # H6908), anti-MYC polyclonal (Sigma cat # C3956), anti-SKP1 (BD Biosciences cat # BDB610530), anti-CUL1 (Life Technologies cat # 71–8700), anti-αTubulin (Sigma cat # T5168), anti-βactin (Sigma cat # A1978), anti-AFG3L2[N1N2] (GeneTex cat # GTX102036), and CRY1-CT and CRY2-CT as described (Lamia et al., 2011).