Hsc70

The interaction of LAMP2A and HSC70 with IFNAR1 was examined by co-immunoprecipitation using a protocol described previously with slight modifications [30 (link)]. Huh-7.5 cells were cultured at 90% confluence in a 100mm tissue culture dish. After 12 hours of serum starvation, cells were washed once with 10ml of PBS and harvested in 3ml of ice-cold RIPA buffer (0.15 mM NaCl, 0.05mM tris-HCl, pH.7.5, 1% triton, 0.1% SDS, 0.1% NP-40 and 1X protease and phosphatase inhibitor cocktail) on a shaker for 30 minutes. One mg of total extract was immunoprecipitated with antibodies (IFNAR1 1:1000 or IFNLR1:1000) overnight at 4°C on a shaker. Approximately 20μl of protein A/G plus-agarose (Santa Cruz Biotechnology) was added at 4°C for one hour. After this step the beads were washed three times with washing buffer (RIPA buffer). The pellet was suspended in 8ul of SDS-PAGE loading buffer and the samples were boiled for 5 minutes. The supernatant was collected and 20μl of lysate were loaded onto 12% acrylamide gene electrophoresis and Western blotting was performed using antibodies to LAMP2A (Abcam), Interferon-alpha receptor-1 (IFNAR1) (Santa Cruz), Interferon-lambda receptor-1 (IFNLR1) (Sigma) and HSC70 (Cell Signaling) as described before.

Western blotting was performed using a standard protocol established in our laboratory. Infected Huh-7.5 cells were washed twice with PBS and then lysed in ice-cold RIPA buffer. Total protein content of the extract was quantified using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of proteins were loaded on SDS-PAGE gels and Western blotting was carried out using antibodies to STAT1, p-STAT1, STAT2, p-STAT2, HSC70, β-actin and GAPDH (Cell Signaling Danvers, MA); as well as LAMP2A (Abcam).

Cells were washed, incubated with Triton Lysis Buffer for 15 minutes and collected. After centrifuging 15 min at 10.000 g pellet and supernatant were separated. Samples were analysed by SDS-page and westernblotting using antibodies recognizing vimentin (D21H3) (rabbit, Cell signaling) and Hsc70 (rabbit, Cell signaling).