Fig. 5

HSP27 interacts with JAK2/STAT5. a The binding of recombinant JAK2 and STAT5a/b at 125 nM to immobilized biotinylated HSP27 was determined by biolayer interferometry Crystallin alpha B and the CBP RING domain serves as a positive control and negative control, respectively (Supplementary Fig. 4a-c) (n=3 independent experiments). b Immunoprecipitation from HEL92.1.7 cell extracts of endogenous HSP27 was followed by immunodetection of endogenous STAT5 and JAK2. CTL: unstimulated cells starved for 16 h; +SVF: Cells starved for 16 h and then stimulated with SVF (10%) for 30 min. Inputs: proteins in total cell lysates. IP IgG: immunoprecipitation with a non-relevant antibody (IgG mouse) (n=2 independent experiments). Uncropped blots presented in Supplementary Fig. 8d. c Immunoprecipitation of endogenous STAT5 was followed by immunodetection of endogenous HSP27 and JAK2. Inputs: proteins in total cell lysates. IP IgG: immunoprecipitation with a non-relevant antibody (IgG mouse) (n=2 independent experiments). Uncropped blots presented in Supplementary Fig. 8e. d Immunofluorescence analysis of the endogenous interaction (red foci) of JAK2 (upper panel) or STAT5 (middle panel) with HSP27 visualized in situ by PLA in HEL92.1.7 cells transfected or not (NT) with a HSP27 siRNA. Nuclei are stained with DAPI. Images were taken randomly and obtained using an Axio Imager 2 at ×40 magnification and analysed using ICY software. Cells were segmented manually, and the number of interaction foci in each cell was counted using the spot detector plugin. Right panel, graphs represent quantification of the interaction of JAK2 or STAT5 with HSP27 visualized in situ by PLA. Each data point corresponds to an analysed cell, placed according to the number of detected interaction foci (lower panel). Immunofluorescence analysis of the endogenous interaction (red foci) of JAK2 with STAT5 visualized in situ by PLA in HEL92.1.7 cells transfected with a scramble (CTL) or a HSP27 siRNA (siHSP27). Nuclei are stained with DAPI. Images were obtained using an Axio Imager 2 at ×63 magnification and analysed using ICY software as in upper panel. Right panel, graphs represent quantification of the interaction of endogenous JAK2 with STAT5 visualized by PLA. P values were calculated using the Mann–Whitney test. ****P < .0001. Scale bar 10 µm