Hap035000

To directly detect the enriched overall ubiquitinated or K48-ubiqutinated ER alpha from the cell extracts, HEK293 cells were transfected with 4 ug Ub or 4 ug K48 Ubi plasmid, 2 ug ER alpha together with 0.5 ug Myc-ZNF213 or Myc-vector. After 48 h, total protein was extracted and pre-cleared with 20ul protein A (santa cruz, SC-2001) for 2 h. The supernatant was collected and immunoprecipitated by ER alpha antibody. Western blot with HA antibody was performed to detect total and K48 poly-ubiquitinated ER alpha.
Immuno uorescence assay MCF-7 cells were xed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-ZNF213 (HAP035000, Sigma) and mouse anti-ERα monoclonal antibodies (SC-56833) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions ful lling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.

To directly detect the enriched overall ubiquitinated or K48-ubiqutinated ER alpha from the cell extracts, HEK293 cells were transfected with 4 ug Ub or 4 ug K48 Ubi plasmid, 2 ug ER alpha together with 0.5 ug Myc-ZNF213 or Myc-vector. After 48 h, total protein was extracted and pre-cleared with 20ul protein A (santacruz, SC-2001) for 2 h. The supernatant was collected and immunoprecipitated by ER alpha antibody. Western blot with HA antibody was performed to detect total and K48 poly-ubiquitinated ER alpha.
Immuno uorescence assay MCF-7 cells were xed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-ZNF213 (HAP035000, Sigma) and mouse anti-ER alpha monoclonal antibodies (SC-56833) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions ful lling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using Image J.