Fig. 4: HSC70 competes with LAMP2A to bind to PARK7.

a Table of top 20 proteins and LAMP2A from Co-IP mass spectrum. b Co-IP analysis of PARK7 and HSC70 in HEK293T cells co-expressing V5-Park7 and HA-Flag-Hsc70. c Colocalization analysis of PARK7 and HSC70 at 37 °C and 39 °C in HEK293T cells co-expressing HA-Flag-Hsc70 and Flag-Park7. d Quantitative analysis of fluorescence lifetime imaging microscopy (FLIM) of CFP fluorescence in HEK293T cells co-expressing CFP-Park7 and YFP-Hsc70 (n = 75). e Endogenous co-immunoprecipitation analysis of PARK7 and HSC70 in BAT from mice housed at RT and TN. f Multiple electrostatic interaction groups are involved in the surface of PARK7/HSC70 complex to maintain the L-shape interface according to the electrostatic properties of the residues. Residues of HSC70 involved were indicated and colored in blue as residues of PARK7 involved were colored in red, respectively. g Co-IP assays of PARK7 mutants M17A, R48A and E15AE18A and HSC70 in HEK293T cells co-expressing Flag-Park7 WT/mutants and HA-Flag-Hsc70. h Immunoblotting for PGC1α and PARK7 protein levels in primary brown adipocytes when overexpressing Park7 mutants in 39 °C for 24 h (representative of n = 3 independent experiments). i Colocalization analysis of PARK7 mutants R48A and E15AE18A and HSC70 at 39 °C in HEK293T cells. Scale bar, 5 µm. j Co-IP analysis of the competitive interaction among PARK7, LAMP2A and HSC70 in HEK293T cells. Cell lysates from HEK293T cells transfected with 2 μg HSC70 and 2 μg LAMP2A with and without different amount PARK7 were used for IP and IB assays. Statistical significance was assessed by unpaired Student’s t-test. All results were expressed as means ± SEM and ****p < 0.001. Student’s two-tailed unpaired T-test for 2-group comparisons. Source data are provided as a Source Data file.