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. Author manuscript; available in PMC: 2020 Mar 3.
Published in final edited form as: Cell Rep. 2019 Feb 12;26(7):1800–1814.e5. doi: 10.1016/j.celrep.2019.01.075

Figure 3. NS2 and MARCH8 Bind and Colocalize with Each Other and Are Localized to the ER.

Figure 3.

(A) IPs with anti-NS2 antibody or IgG control from membrane fractions derived from Huh7.5.1 cells 72 hr after transfection with HCV RNA. Membranes were blotted with either anti-MARCH8 (top left) antibody or neutralized anti-MARCH8 antibody (after preincubation with recombinant MARCH8; top middle), and antibodies against MARCH10, NS2, and actin. Molecular weight markers are indicated on the right (kDa). Blotting with the neutralized anti-MARCH8 antibody revealed that only the 33-kDa (but not the 45- and 50-kDa) bands correspond to MARCH8. IP, immunoprecipitation; IB, immunoblotting; WCL, whole cell lysates.

(B) IPs with anti-FLAG antibody or IgG control from membrane fractions derived from 293T cells ectopically expressing FLAG-MARCH8 and/or GLuc-NS2. Membranes were blotted with antibodies against GLuc, FLAG, and actin.

(C–E) Confocal IF microscopy images of MARCH8 (red) and NS2 (blue) (C), MARCH8 (red) and ER (green) (D), and all three components (E) in naive and HCV (J6/JFH) RNA-transfected Huh7.5.1 cells ectopically expressing FLAG-MARCH8 48 hr after transfection. Shown are representative merged images at 60× magnification and quantitative data (means ± SD; **p < 0.01, ***p < 0.001 relative to naive control). n = ~20 cells per category. The arrows indicate MARCH8-NS2 colocalization in the ER.