Figure 1
Figure 1. Release of cellular HJV. (A) Analysis of cell-associated and secreted HJV after blocking protein synthesis. HJV-HepG2 cells in 12-well plates were incubated in 400 μL complete medium with 100 μg/mL cycloheximide (CHX) to block protein synthesis. After 0, 1, 2, 4, and 6 hours of incubation, conditioned medium (CM) was collected, and cell lysate (L) was prepared using 100 μL NET-Triton buffer (150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 10 mM Tris, pH 7.4, and 1% Triton X-100) with 1× Protease inhibitors cocktail (Roche Diagnostics, Indianapolis, IN). Each time point was performed in duplicate. Western blotting was performed on the total lysates and one-third of conditioned medium using rabbit anti-HJV (0.22 μg/mL) and mouse anti–beta-actin (1:10 000) (lysates only). Control-HepG2 cells (Ctrl) were used as a negative control. HJV is approximately 50 kDa in the cell lysates and 38 kDa in the CM. β-Actin was used as a loading control for the cell lysates. (B) Pulse-chase analysis of cellular HJV. Cells were metabolically labeled with 35S-(Met/Cys) (PerkinElmer Life and Analytical Sciences, Waltham, MA) at 100 μCi/mL in minimum essential medium without met/cys for 30 minutes, washed and then incubated in regular growth medium for 0, 0.5, 1, 2, 3, 4, 5, and 6 hours. Cell lysates were then collected and immunoprecipiated using rabbit anti-HJV 18745 antibody. Immunoprecipiated proteins were washed and separated by SDS-PAGE. Image was obtained by exposure to x-ray film. (C) Analysis of cellular and secreted HJV after induction of HJV synthesis. Expression of HJV in tTA-HJV-HepG2 was induced by addition of 2 μg/mL doxycycline into the culture medium. The entire cell lysate (L) and one-third of CM after 0, 1, 2, 4, 6, 8, and 10 hours were subjected to Western blots for HJV in cell lysates and medium and β-actin in lysates. Dox indicates doxycycline treated. (D) Contribution of lysosomal degradation to HJV turnover. HJV-HepG2 cells in 12-well plates were incubated in presence of 100 μg/mL cycloheximide with or without addition of 100 nM bafilomycin A (Baf) for 0, 1, 2, 3, 4, 5, and 6 hours. Proteins from whole-cell lysates (L) and media (CM) precipitated with 6% trichloroacetic acid (TCA) were subjected to immunodetection. Experiments were repeated 3 times with consistent results.

Release of cellular HJV. (A) Analysis of cell-associated and secreted HJV after blocking protein synthesis. HJV-HepG2 cells in 12-well plates were incubated in 400 μL complete medium with 100 μg/mL cycloheximide (CHX) to block protein synthesis. After 0, 1, 2, 4, and 6 hours of incubation, conditioned medium (CM) was collected, and cell lysate (L) was prepared using 100 μL NET-Triton buffer (150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 10 mM Tris, pH 7.4, and 1% Triton X-100) with 1× Protease inhibitors cocktail (Roche Diagnostics, Indianapolis, IN). Each time point was performed in duplicate. Western blotting was performed on the total lysates and one-third of conditioned medium using rabbit anti-HJV (0.22 μg/mL) and mouse anti–beta-actin (1:10 000) (lysates only). Control-HepG2 cells (Ctrl) were used as a negative control. HJV is approximately 50 kDa in the cell lysates and 38 kDa in the CM. β-Actin was used as a loading control for the cell lysates. (B) Pulse-chase analysis of cellular HJV. Cells were metabolically labeled with 35S-(Met/Cys) (PerkinElmer Life and Analytical Sciences, Waltham, MA) at 100 μCi/mL in minimum essential medium without met/cys for 30 minutes, washed and then incubated in regular growth medium for 0, 0.5, 1, 2, 3, 4, 5, and 6 hours. Cell lysates were then collected and immunoprecipiated using rabbit anti-HJV 18745 antibody. Immunoprecipiated proteins were washed and separated by SDS-PAGE. Image was obtained by exposure to x-ray film. (C) Analysis of cellular and secreted HJV after induction of HJV synthesis. Expression of HJV in tTA-HJV-HepG2 was induced by addition of 2 μg/mL doxycycline into the culture medium. The entire cell lysate (L) and one-third of CM after 0, 1, 2, 4, 6, 8, and 10 hours were subjected to Western blots for HJV in cell lysates and medium and β-actin in lysates. Dox indicates doxycycline treated. (D) Contribution of lysosomal degradation to HJV turnover. HJV-HepG2 cells in 12-well plates were incubated in presence of 100 μg/mL cycloheximide with or without addition of 100 nM bafilomycin A (Baf) for 0, 1, 2, 3, 4, 5, and 6 hours. Proteins from whole-cell lysates (L) and media (CM) precipitated with 6% trichloroacetic acid (TCA) were subjected to immunodetection. Experiments were repeated 3 times with consistent results.

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