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. 2019 Feb 21;294(16):6375–6386. doi: 10.1074/jbc.RA118.006263

Figure 3.

Figure 3.

Knockdown of CASPR1 reduced the glycosylation and plasma membrane localization of ATP1B3 in HBMECs. A, CASPR1-specific shRNA was stably transfected to HBMECs. Then the cell lysates were analyzed by Western blotting using CASPR1 and ATP1B3 (ab67409) antibody, with GAPDH as loading control. An asterisk represents the fully glycosylated ATP1B3, a rhombus indicates the intermediately forms of ATP1B3, and a circle indicates the core proteins of ATP1B3 (left). The knockdown effect was calculated as the band density of CASPR1 divided by that of GAPDH (right). The change of the fully glycosylated, intermediately glycosylated, and core protein of ATP1B3 was calculated as the band density of three forms of ATP1B3 divided by that of GAPDH (right). Values are mean ± S.D. (error bars) from three independent experiments. **, p < 0.01 (Student's t test). B, HBMECs with CASPR1 knockdown were immunostained with antibodies recognizing ATP1B3 (ab137055, red) and CASPR1 (ab34151, green). DAPI (blue) was used for counterstaining. Arrows, plasma membrane localization of ATP1B3. Images are representative of three independent experiments. Scale bar, 20 μm. C, HBMECs with CASPR1 knockdown were incubated with medium containing Sulf-NHS-LC-biotin for 30 min to label the plasma membrane protein, and then the cell lysates were immunoprecipitated (IP) with ATP1B3 antibody (H00000483-D01). The precipitated proteins were analyzed by Western blotting (WB) with ATP1B3 antibody (ab67409) as well as horseradish peroxidase–conjugated streptavidin to recognize the biotin-labeled proteins. The total cell lysates (TCL) were analyzed by Western blotting with ATP1B3 antibody (ab67409), with GAPDH as loading control (left). The percentage of plasma membrane ATP1B3 was calculated as the band density of biotin-labeled ATP1B3 divided by that of precipitated ATP1B3 (right). Values are mean ± S.D. from three independent experiments. **, p < 0.01 (Student's t test). D and E, for rescue experiments, the HBMECs with CASPR1 knockdown were transfected with constructs encoding GFP-tagged CASPR1, with the empty vector (GFP alone) serving as control. The transfected cells were lysed and analyzed by Western blotting with ATP1B3, with GAPDH as loading control (D). As indicated, the transfected cells were analyzed by immunofluorescence with ATP1B3 antibody (ab137055, red) and GFP (green). DAPI (blue) was used for counterstaining (E). Images are representative of three independent experiments. Scale bar, 20 μm.