Hap043396

To directly detect the enriched K48-ubiquitinated and total ubiquitinatoin P53 from the cell extracts, HEK293 cells were transfected with 0.8 ug K48 Ubi or 0.8 ug Ub plasmids together with 0.8 ug GFP-P53 plasmid and 0.8 ug Myc-TRIM3 or Myc-vector. After 24 h, the cells were treated with 20 uM MG132 for 7 hours,then total protein was extracted and pre-cleared with 30ul protein A (santa cruz, SC-2001) for 4 h.
The supernatant was collected and immunoprecipitated by P53 antibody. Western blot with HA antibody was performed to detect total or K48 poly-ubiquitinated P53.
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-TRIM3 polyclonal antibody (HAP043396, Sigma) and mouse anti-P53 monoclonal antibodies (SC-126, Santa Cruz) 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.

MCF-7 cells were fixed 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-TRIM3 polyclonal antibody (HAP043396, Sigma) and mouse anti-P53 monoclonal antibodies (SC-126, Santa Cruz) 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 fulfilling 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 K48-ubiquitinated and total ubiquitinatoin P53 from the cell extracts, HEK293 cells were transfected with 0.8 ug K48 Ubi or 0.8 ug Ub plasmids together with 0.8 ug GFP-P53 plasmid and 0.8 ug Myc-TRIM3 or Myc-vector. After 24 h, the cells were treated with 20 uM MG132 for 7 hours,then total protein was extracted and pre-cleared with 30ul protein A (santa cruz, SC-2001) for 4 h.
The supernatant was collected and immunoprecipitated by P53 antibody. Western blot with HA antibody was performed to detect total or K48 poly-ubiquitinated P53.
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-TRIM3 polyclonal antibody (HAP043396, Sigma) and mouse anti-P53 monoclonal antibodies (SC-126, Santa Cruz) 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, K63-ubiquitinated, mono-ubiquitinated and K48-ubiqutinated ER alpha from the cell extracts, HEK293 cells were transfected with 4 ug Ub, 4 ug K63 Ubi, 4 ug Ub-KO or 4 ug K48 Ubi plasmid, 2 ug ER alpha together with 0.5 ug Myc-TRIM3 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 specific ubiquitinated ER alpha. Immunofluorescence assay MCF-7 cells were fixed 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-TRIM3 (HAP043396, 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 fulfilling 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.