Goat anti mouse igg conjugated to irdye800cw

After the indicated treatments, the cells were washed three times with PBS. The cell lysates were prepared via the addition of SDS-PAGE sample reducing buffer, followed by heating at 95°C for 10 min. Immunoblotting was performed using anti-phospho ERK1/2 (D13.14.4E) (1:1,000 dilution), anti-ERK1/2 (137F5) (1:1,000 dilution) and anti-IκBα alpha (L35A5) (1:2,000 dilution) antibodies from Cell Signaling Technology. An anti-actin antibody (Millipore) (1:5,000 dilution) was used to confirm equal loading. The secondary antibodies included goat anti-rabbit IgG and goat anti-mouse IgG conjugated to IRdye800CW (Li-COR) (1:10,000 dilution). ImageJ software was used to quantify band intensities, and actin was used to normalize the total amount of protein loaded in each well.

The method used for cell lysate preparation and immunoblotting were described previously (Pathak et al., 2013 (link)). Briefly, cell lysates were prepared by the addition of SDS-PAGE sample reducing buffer to cells after washing twice with PBS, followed by heating at 95°C for 10 min. Immunoblotting was performed using anti-phospho ERK1/2 (D13.14.4E) (1:1,000 dilution), anti-phospho-p38 (D3F9), anti-phospho-JNK (81E11), and anti-ERK1/2 (137F5) (1:1,000 dilution) antibodies from Cell Signaling Technology. Anti-phospho-c-Fos (Thr325) antibody was from Bioss antibodies. An anti-actin antibody (Thermo Scientific) (1:5,000 dilution) was used to confirm equal loading. The secondary antibodies included goat anti-rabbit IgG and goat anti-mouse IgG conjugated to IRdye800CW (Li-COR) (1:10,000 dilution). Band intensities were quantified using the ImageJ software, and actin was used to normalize the total amount of protein loaded in each well.

Reduced samples were separated using 10% SDS-PAGE and transferred to PVDF membranes (Immobilon-FL, Millipore-Sigma). Membranes were blocked with Odyssey Blocking Buffer for 20 min, after which primary antibodies (at concentrations recommended by the manufacturers) in PBS with 5% BSA, 0.05% Tween 20 were added and incubated overnight at 4 °C. Primary antibodies were as follows: affinity-purified polyclonal antibody for the N terminus of YRS (no. A305-064A, Bethyl Laboratories); monoclonal antibodies phospho-NF-κB p65 (Ser-536; no. 3033), and total IκB-α (no. 4814) (both Cell Signaling Technology); α-tubulin (no. sc-53646, Santa Cruz Biotechnology). After washing the membranes in PBS with 0.05% Tween 20, secondary antibodies were incubated (at concentrations recommended by manufacturers) in PBS with 1% BSA, 0.05% Tween 20 for 1 h at 25 °C. Secondary antibodies were as follows: goat anti-mouse IgG conjugated to IRDye 800CW (LI-COR) and goat anti-rabbit IgG conjugated to Alexa Fluor® 680 (Thermo Fisher Scientific). Immunoblots were washed and analyzed using a LI-COR Odyssey IR imager, and immunoreactive bands were quantified by densitometry using ImageJ software (National Institutes of Health).