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. 2021 Feb 23;22(4):2207. doi: 10.3390/ijms22042207

Figure 6.

Figure 6

NKCC2 interacts with the stress-inducible Hsp70. (A) Hsp70 interacts also with immature NKCC2. Cell lysates from OKP cells transiently transfected with Myc-NKCC2 singly or in combination with of Myc-Hsp70 were immunoprecipitated (IP) with anti-Hsp70 or anti-V5 antibody. NKCC2 protein was recovered from Hsp70 immunoprecipitates only in its immature form (lane 3). (B) Similar to STCH, Hsp70, and NKCC2 co-localizes at the ER. Upper panel, immunofluorescence confocal microscopy showing distribution of NKCC2 and Hsp70 in HEK cells. Transiently transfected HEK cells with EGFP-NKCC2 and Myc-HSP70, were fixed, permeabilized, and then stained with mouse anti-Myc for NKCC2 (Texas Red). The yellow color (merged image) illustrates co-localization of the proteins. Middle panel, HEK cells transfected with Myc-Hsp70 were stained with mouse anti-Myc (Texas Red; red) and rabbit anti-calnexin (FITC; green). Yellow indicates overlap between Hsp70 (red) and the ER marker (green). Lower panel, STCH colocalizes with Hsp70. HEK cells transiently transfected with GFP-STCH and Myc-Hsp70, were fixed and permeabilized before being stained with mouse anti-Myc for Hsp70 (Texas red, Red). Yellow illustrates overlap between Hsp70 (red) and the STCH (green). Bars, 5 μm.