Phospho nf κb p65 rabbit mab

The kidney tissues were cut into 5-μm-thick slices on a cryostat and stored at −20°C until use [36 (link)]. The tissue sections were blocked with 1% bull serum albumin for 1 h at room temperature, and then incubated overnight with phospho-NF-κB p65 rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Abcam, Cambridge, UK). The DAPI staining was conducted according to the experimental protocol. Morphological analyses were performed by using AX10 imager A2/AX10 cam HRC fluorescence microscope (Zeiss, Jena, Germany).

Cells were treated in α-MEM containing 2 μg/mL Hlb for the indicated minutes. To extract total cell protein, cells were harvested after washing with cold PBS and lysed with cold RIPA lysis buffer containing protease and phosphatase inhibitors. The samples were resolved on a 12% SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF) membranes. Anti-STAT5A Ab, phospho-STAT5A Ab (BBI Life Sciences), phospho-STAT4 Ab, STAT4 rabbit mAb, NF-κB p65 rabbit mAb, phospho-NF-κB p65 rabbit mAb, p44/42 MAPK rabbit mAb, phospho-p44/42 MAPK rabbit mAb (Cell Signaling Technology), anti-GAPDH, and horseradish peroxidase-conjugated goat antibodies against rabbit IgG and mouse IgG (Jackson ImmunoResearch Laboratories) were used. Finally, the blots were detected using Pierce™ ECL Western Blotting Substrate (Thermo).