Mouse monoclonal anti v5 antibody

Approximately 50,000 HeLa cells were seeded in Nunc™ Lab-Tek™ II Chamber Slide™ System Thermo Scientific™ and transfected 24 hours later with 1 µg of plasmid DNA according to the Fugene^® protocol. Two days after transfection, coverslip grown transfected HeLa cells were washed three times with PBS and fixed with 4% PFA for 15 min. Cells were then washed five times and permeabilized with a 50% methanol/acetone mix for 10 min. After five PBS washings, permeabilized cells were incubated for 1 hour at room temperature, first with 1% bovine serum albumin (BSA) PBS 1/200 mouse monoclonal anti-V5 antibody (Invitrogen) and then with 1% BSA PBS 1/750 anti mouse Alexa Fluor 488 conjugated antibody. After several PBS washings, coverslips were mounted with Mounting medium for immunofluorescence (Interchim). Imaging was performed using a Leica SP5 confocal microscope. Colocalization analyses were performed with Huygens Essential for Mac version 4.3.1p3 (Scientific Volume Imaging B.V.). Images were first deconvoluted then analyzed using the Costes method.

HEK-293 cells expressing the BKαγ1^WT or BKαγ1^F273S channel complexes were preincubated with 2 μM mallotoxin in PBS buffer (pH 7.4) for 5 minutes. The channel complexes were then solubilized from cell membranes with 1% dodecyl maltoside (DDM) in Tris-buffered saline (TBS) buffer (50 mM Tris, 150 mM NaCl, pH 7.6) supplemented with 2 μM mallotoxin. After centrifugation at 17,000 g for 10 minutes, the solubilized channel complexes in the supernatant were incubated for 2 hours with mouse monoclonal anti-BKα antibody (University of California–Davis/NIH Neuromab facility) that was covalently crosslinked to protein-A/G agarose beads (Thermo Fisher Scientific). The captured protein complexes were washed three times with TBS buffer supplemented with 1% DDM and 2 μM mallotoxin, eluted with SDS-PAGE sample buffer, and then loaded directly to 12% SDS-PAGE gel to be separated by electrophoresis. Resolved proteins were transferred to PVDF membranes (Thermo Fisher Scientific) and probed by a mouse monoclonal anti-V5 antibody (1:10000, Invitrogen) for V5-tagged γ subunits and a mouse monoclonal anti-BKα antibody (1:1000, University of California–Davis) for BKα. The intensities of the protein bands were analyzed with ImageJ software (US National Institutes of Health).

Expression plasmids for human PERK and its DN mutant K621M were obtained from Ronald Wek [48 (link)]. Reporter plasmid pCHOP-Luc, in which luciferase expression is driven by human CHOP promoter (-644 to +91), was provided by Nai Sum Wong [23 (link)]. Reporter plasmids pGRP78-Luc and pGRP94-Luc were gifts from Kazutoshi Mori [40 (link),58 (link)]. The Grp78 and Grp94 promoters are derived from -304 to +34 of human Grp78 gene and -363 to +34 of human Grp94 gene, respectively. Both promoters harbor multiple copies of ER stress response element [58 (link)]. pUPRE-Luc reporter plasmid has been described elsewhere [41 (link),42 (link)].
Mouse monoclonal anti-V5 antibody was purchased from Invitrogen. Mouse anti-FLAG antibody (clone M2) was from Sigma-Aldrich.