Anti nrf2 sc 365949

Pancreas and lung sections were incubated with the following primary antibodies: Anti-NRF2 (sc-365949, 1:200, Santa Cruz Biotechnology, CA, USA); anti-HO-1 (sc-136960, 1:200, Santa Cruz Biotechnology, CA, USA); anti-Mn-SOD (sc-137254, 1:200, Santa Cruz Biotechnology, CA, USA); anti-NLRP3 (sc-134306, 1:200, Santa Cruz Biotechnology, CA, USA); anti-Caspase-1 (sc-56036, 1:200, Santa Cruz Biotechnology, CA, USA); and anti-ASC (sc-514414, 1:200, Santa Cruz Biotechnology, CA, USA), as previously described [48 (link)]. Sections were then incubated with the following secondary antibodies: Peroxidase-conjugated bovine anti-mouse immunoglobulin G (IgG) or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research, West Grove, PA, USA). Specific marking was revealed with a biotin-conjugated goat anti-rabbit IgG or biotin-conjugated goat anti-mouse IgG and avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Graphic presentation of densitometric analyses was performed Image J software (v1.52a) as previously described [49 (link)]. All immunohistochemical analyses were conducted by an observer without knowledge of the treatments.

The cellular lysis was performed using an SDS denaturing lysis solution in which the protease inhibitor cocktail (PIC), phenylmethane sulfonyl fluoride (PMSF), Na₃VO₄ and β-mercaptoethanol were added. After SDS/PAGE electrophoresis proteins were transferred to a 0.2 µm-pore-sized nitrocellulose membrane. Immunoblotting was performed as previously described^{11 (link)}. The used primary antibodies were: anti-PP2Ac (Upstate, 05-545); anti-actin (Sigma-Aldrich, A2066); anti-CAPN1 (sc-271313), anti-GSK3β (sc-377213) and anti-NRF2 (sc-365949) (Santa Cruz Biotechnology); anti-p-GSK3β S9 (9336), anti-p-Akt T308 (4056); anti-p-Akt T473 (9271), anti-Akt (9272), anti-Caspase-3 (9662) (Cell Signaling Technology), anti-Smac/DIABLO^{39 (link)} and anti-HDAC4^{66 (link)}; anti-Caspase-2^{67 (link)}; anti-tubulin^{67 (link)}. The same membranes were incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The used secondary antibodies were goat anti-mouse or goat anti-rabbit (Sigma Aldrich). Finally, the blots were developed using Super Signal West Dura as recommended by the vendor (Pierce Waltham, MA, USA).

The following antibodies were used: anti-β-actin, anti-G6PD (Sigma-Aldrich); anti-HBcAg (Abcam, Cambridge, MA, USA), anti-Keap1 (Cell Signaling, Danvers, MA, USA); anti-Myc, anti-Nrf2 (sc-365949), anti-Lamin B, anti-p62, and anti-Flag (Santa Cruz, Santa Cruz, CA, USA); anti-GFP (BD Biosciences, Franklin Lakes, NJ, USA); goat anti-rabbit IRDye800CW and goat anti-mouse IRDye680 (LI-COR Biosciences, Lincoln, NE, USA). Plasmids with the 1.3mer HBV genomic DNA (pUC19 HBV), the HBx-negative 1.3mer HBV genomic DNA (pUC19 HBVX⁻) and HBx-GFP were described previously.^{19 (link), 36 (link)} Dsred-Keap1 was provided by Yue Xiong (University of North Carolina at Chapel Hill, Chapel Hill, NC, USA). For Flag-p62 construction, the p62 coding region was amplified from Cherry-GFP-p62, a gift from Terje Johansen (University of Tromsø, Tromsø, Norway), and was inserted into pCMV-Tag 2A vector using the EcoRI and XhoI restriction sites. HBx-Myc was made by cloning the cDNA of HBx into pcDNA-3.1 vector using EcoRI and XhoI restriction sites. The point mutation for Flag-p62 T350A and Dsred-Keap1 N382A was created by PCR-based site-directed mutagenesis using primer: 5′-AAGAAGTGGACCCGTCTGCAGGTGAACTCCAGTCC-3′ (p62-sense), 5′-GGACTGGAGTTCACCTGCAGACGGGTCCACTTCTT-3′ (p62-antisense), 5′-CGTGGGCGGCAGGGCCAACTCGCCCGAC-3′ (Keap1-sense) and 5′-GTCGGGCGAGTTGGCCCTGCCGCCCACG-3′ (Keap1-antisense).