Figure 2. MGRN1 interacts with Hsp70.

(A–B) Transfection was used to overexpress MGRN1-Myc and Hsp70-EGFP in Cos-7 cells. Cells were collected, lysed, and processed for immunoprecipitation (IP) with an MGRN1 antibody 24 hrs post-transfection. Blots were probed with an Hsp70 (A) antibody and Myc (B) antibody. (C–D) As described in A, the samples were processed for IP using an Hsp70 antibody and the blots were probed with anti-MGRN1 (C) and anti-GFP (D). (E) Hsp70-EGFP transfected cell lysate were pulled down by MGRN1 antibody and blot was developed with GFP antibody. (F) As described in section A-E; same samples were pulled down by beads only (control) and blots were obtained with anti-MGRN1, anti-Hsp70 and anti-GFP antibodies. (G) The cells were transfected as described in A with MGRN1-GFP plasmid and 36 hrs of post transfection some cells pretreated with 50 nM Bafilomycin (Baf) and exposed to 43°C for 30 minutes heat shock (HS). Immediately after heat shock treatment cells were processed to immunofluorescence staining using Hsp70 antibody. Rhodamine-conjugated secondary antibody was used to stain Hsp70 protein. Arrows indicate the recruitment of MGRN1 to the induced Hsp70 inclusions.