Generation of lentivirus(27 (link)): HEK-293T cells (5 x 106) were transfected with the viral packaging plasmids: p-CMV-VSV-G, pHR’8.2ΔR, and NHERF1 shRNA (Sigma, TRCN0000043736 for NHERF1) or scrambled-sequence control shRNA (Sigma, SCH002) using the Lipofectamine® 2000 reagent (Invitrogen). Viral supernatants were harvested 72 h post-transfection, 0.45 μm sterile-filtered, and concentrated using Vivaspin™ protein concentrators (100 kDa MWCO, GE Healthcare).
Lentiviral transduction(27 (link)): LAD2 and RBL-2H3 cells (5 x 106) were washed twice and plated in complete media with hexadimethrine bromide (polybrene, Sigma-Aldrich, 4 μg/mL). Concentrated viral supernatant was then added to cells, centrifuged at 700g for 1 h, and incubated for 8-10 h in 37°C and 5% CO2. Media was changed and cells were exposed to puromycin (2 μg/mL) for selection of stable clones and viable cells were used for Ca2+ mobilization experiments.
Lentiviral transduction(27 (link)): LAD2 and RBL-2H3 cells (5 x 106) were washed twice and plated in complete media with hexadimethrine bromide (polybrene, Sigma-Aldrich, 4 μg/mL). Concentrated viral supernatant was then added to cells, centrifuged at 700g for 1 h, and incubated for 8-10 h in 37°C and 5% CO2. Media was changed and cells were exposed to puromycin (2 μg/mL) for selection of stable clones and viable cells were used for Ca2+ mobilization experiments.
