Femto enhanced chemiluminescence ecl substrate

Lysates were analyzed by SDS-PAGE (NuPAGE 4-to-12% bis-Tris gel; Novex Life Technologies) and electrophoretically transferred to a nitrocellulose membrane (Novex Life Technologies). Immunoblot membranes were cut at multiple molecular weights to allow for multiple analytes to be probed within individual experiments. The membrane was blocked with 5% bovine serum albumin (BSA) in PBST (1× PBS and 1% Tween 20) and probed overnight at 4 °C with primary antibodies detailed in supplemental table 1. The following day, membranes were washed three times with PBST and incubated with goat anti-mouse horseradish peroxidase (HRP)-conjugated and goat anti-rabbit HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch) in 5% BSA in PBST for 1 h at room temperature. Blots were developed in SuperSignal West Dura, or Femto-enhanced chemiluminescence (ECL) substrate (Thermo Scientific). Relative intensity was quantified by densitometry using ImageJ and normalized to the intensity of GAPDH or HSP90. Housekeeping probes were chosen to minimize interference with proteins of interest, after confirming that Gucy2c^−/− expresses comparable levels of HSP90 as
Gucy2c^+/+ (supplemental Fig. 2e, f). All blots were processed in parallel and derived from the same experiments.

Lysates were analyzed by SDS-PAGE (NuPAGE 4-to-12% bis-Tris gel; Novex Life Technologies) and electrophoretically transferred to a nitrocellulose membrane (Novex Life Technologies). The membrane was blocked with 5% bovine serum albumin (BSA) in PBST (1× PBS and 1% Tween 20) and probed overnight at 4°C with primary antibodies detailed in Supplemental table 1. The following day, membranes were washed three times with PBST and incubated with goat anti-mouse horseradish peroxidase (HRP)-conjugated and goat anti-rabbit HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch) in 5% BSA in PBST for 1 h at room temperature. Blots were developed in SuperSignal West Dura or Femto enhanced chemiluminescence (ECL) substrate (Thermo Scientific). Relative intensity was quantified by densitometry using ImageJ and normalized to the intensity of GAPDH or HSP90. Housekeeping probes were chosen to minimize interference with proteins of interest, after confirming that Gucy2c^−/− express comparable levels of HSP90 as Gucy2c^+/+ (Supplemental fig. 2E–F).