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. 2019 Nov 21;294(52):20196–20206. doi: 10.1074/jbc.RA119.007980

Figure 5.

Figure 5.

Subcellular localization of flotillin-1 phosphomutants. A, immunofluorescent staining of pcDNA3.1/myc-His A-flotillin-1 WT, Gö6976-treated (1 μm, 30 min) WT, and phospho-null (S315A) and phosphomimic (S315D) flotillin-1–transfected BPAECs was performed using a tag-specific c-myc antibody (green). A VE-cadherin (red) antibody was used to detect cell membranes, and nuclei were stained with DAPI (blue). Magnified sections (marked by white rectangles) on merged images show membrane regions of the cells. Scale bars = 25 μm. B, the membrane fraction was isolated from control, flotillin-1 WT and flotillin-1 S315A– and flotillin-1 S315D–transfected BPAECs. Total lysates and membrane fractions were analyzed with anti-c-myc, anti-actin (cytoplasmic marker), and anti-CD31 (membrane marker) antibodies. CTR, control; WB, Western blot. C, membrane and cytoplasmic fractions were made from untreated and Gö6976-treated cells transfected with pcDNA3.1/myc-His A-flotillin-1 WT. Fractions were analyzed by Western blotting.