Rpn2106

Total cell extracts were fractionated by gel electrophoresis. Proteins were transferred to PVDF membranes and immunoblotted using following antibodies: GAPDH (abcam, ab8245), ß-Actin (abcam, ab8226), Met (L41G3, Cell Signaling, 3148) and Phospho-Met (Tyr1234/1235) (Cell signaling, 3126), MAPK (Erk1/2) (06–182, Millipore) and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9101, Cell Signaling). The protein-antibody complexes were detected by horseradish peroxidase-conjugated secondary antibodies incubated with a chemiluminescent reagent (Amersham, RPN2106).

Transfected primary chondrocytes were lysed with ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40 with proteinase and phosphatase inhibitors). 50 μg of protein lysates were run on a 10% SDS-PAGE and transferred to a 0.45 μm PVDF-membrane by semi-dry transfer (PerfectBlue™, PeqLab). The membrane was blocked with 5% milk and incubated with the appropriate primary antibodies anti-GFP (1:15000, Abcam, #ab13970, RRID:AB_300798), anti-Myc (1:1000, Cell Signaling Technology, mAb #2276, RRID:AB_331783), pan-CaMKII (1:1000, Cell Signaling Technology, mAb #4436S, RRID:AB_10545451), anti-Flag (1:1000, Sigma-Aldrich, #F1804, RRID:AB_262044), and proteasome 20 (P20) (1:15000, Abcam, #ab3325, RRID:AB_303706), followed by incubation with the respective, species-specific HRP-coupled secondary antibodies (1:1000 and 1:5000). ECL substrate was used for signal development (Amersham, #RPN2106) on X-ray film (Amersham).

Cell lysis and Western blot analysis were performed as described previously (92 (link)). Briefly, equal amounts of protein (20 μg) were run on SDS–polyacrylamide gel electrophoresis gels, transferred to nitrocellulose membranes, and immunoblotted with the following antibodies: p-eIF2α (1:500; #9721, Cell Signaling Technology, Danvers, MA), p-PERK (1:500; #3191, Cell Signaling), and growth arrest and DNA damage–inducible protein (GADD153)/CHOP (1:500; sc-575, Santa Cruz Biotechnology, Dallas, TX). Following incubation with secondary antibody conjugated to horseradish peroxidase, the respective bands were detected by enhanced chemiluminesence (RPN2106, Amersham Biosciences, Piscataway, NJ). Immunoblots were scanned via Scion Image software.

DENV-containing culture supernatants were obtained from C6/36 cells and divided into two groups. The reducing group was added with loading buffer containing 100 mM beta-mercaptoethanol while the unheated and non-reducing group was added with loading buffer without beta-mercaptoethanol. The samples were then separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (10600023; Amersham) by electroblotting. Non-specific binding was blocked using 5% skimmed milk, after which the membranes were treated with DENV-specific HMAb 1G5 (1 μg/ml) and then horseradish peroxidase-conjugated antibody against human IgG (1:5000, v/v). The membranes were then treated with an enhanced chemiluminescence substrate (RPN2106, Amersham). DV69.6 (1 μg/ml) (Beltramello et al., 2010 (link)), which is a cross-reactive antibody against DENV, recognizes the prM protein and is an indicator of the molecular weight of prM. 4G2 (1 μg/ml) (Henchal et al., 1985 (link)), which is a cross-reactive antibody against flavivirus, recognizes a conserved epitope that is present on the fusion loop of the E protein; it is an indicator of the molecular weight of the E protein.