Ab7977

Spinal sections were stained with free-floating 3,3′-diaminobenzidine tetrahydrochloride (DAB). The sections were rinsed with 0.05 M PBS and incubated in 1% hydrogen peroxide in PBS at room temperature for 15 min. The sections were then incubated with primary antibodies against Bax (1:200, ab7977, Abcam), iNOS (1:200, #610329, BD), COX-2 (1:200, #160106, Cayman), and Iba1 (1:500, #019-19741, Wako) at 4 °C overnight. After washing with PBS three times, they were then incubated with biotinylated anti-rat secondary antibody (1:200, Millipore, Billerica, MA, USA) at room temperature for 1 h. After rinsing three times with PBS, they were subjected to avidin-biotin complex (Vector Laboratories, Burlingame, CA, USA) with peroxidase coupling in a mixture containing 0.05% DAB (Sigma-Aldrich, St. Louis, MO, USA) and 0.03% H₂O₂ for 2–5 min. Counter-staining was performed lightly using 0.2% methyl green solution or 0.1% CV solution. Images of DAB-stained brain sections were captured using a light microscope (BX51, Olympus, Tokyo, Japan) equipped with CCD camera (DP70, Olympus).

Protein expression in DU145 cells treated with either CK (control) or si-NK, astaxanthin or si-STAT3, or astaxanthin and si-STAT3 was evaluated using Western blotting. DU145 cells were seeded in 6-well plates and cultured to 80% confluency. The cells were then treated with CK (control) or si-NK, astaxanthin or si-STAT3, or astaxanthin and si-STAT3 for 24 h, followed by protein extraction. An equivalent of 30 μg protein was subjected to SDS-PAGE and Western blotting. The primary antibodies were diluted as follows: Bcl-2(26 kDa) 1:1000 (ab692, Abcam, Cambrige, MA, USA), Bax (21 kDa) 1:1000 (ab7977, Abcam, Cambrige, MA, USA), Caspase3 (32 kDa) 1:1000 (ab32351, Abcam, Cambrige, MA, USA), Caspase9 (46 kDa) 1:1000 (ab32539, Abcam, Cambrige, MA, USA), NF-κB p65 (65 kDa) 1:1000 (ab76302, Abcam, Cambrige, MA, USA), JAK2 (131 kDa) 1:1000 (ab92552, Abcam, Cambrige, MA, USA) and Stat3 (88 kDa) 1:1000 (ab119352, Abcam, Cambrige, MA, USA). Each Western blotting assay was repeated at least two times.

Collected cells, which were treated with 2.5 and 5 mg/mL of TM for 24 h, and collected tumor tissues were lysed by RIPA buffer (Sigma-Aldrich, USA) containing 1% protease inhibitor cocktail (Sigma-Aldrich, USA) and 2% phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich, USA). Protein concentrations were determined using Bradford method. 40 μg of proteins was separated by 10–12% SDS-PAGE gels and then transferred electrophoretically onto PVDF membranes. The transferred membranes were blocked in 5% bull serum albumin (BSA) for 4 h and then blotted with the following primary antibodies at 4°C overnight at dilution of 1 : 1000: cleaved poly(ADP-ribose) polymerase (cleaved-PARP) (ab32064), Bad (ab129192), Bax (ab7977), B-cell lymphoma-2 (Bcl-2) (ab32124), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245) (Abcam, Cambridge, MA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies diluted at 1 : 2000 (Santa Cruz, USA). ECL detection kits (Millipore, USA) were applied to detect chemiluminescence of blots, and the intensity was quantified by scanning densitometry using Image J (NIH, Bethesda, MD).

Cochlear homogenates were prepared in Laemmli sample buffer. Blots were incubated with antibodies recognizing Sirtuin 3 (SIRT3, 1/1000, Cell Signaling #5490 RRID:AB_10828246), p-Beclin 1 (1/200, Cell Signaling Ser15, # 84966 RRID:AB_2800045), Nrf2 (1/1000, Santa Cruz Biotechnology # sc-365949, RRID:AB_10917561), SOD2 (1/1000, abcam #ab13533, RRID:AB_300434), Catalase (1/1000, sigma Aldrich #SAB4503383, RRID:AB_10747206), LC3B (1/800, Cell Signaling #2775 RRID: AB-915950), Bnip3 (1/1000, Abcam, #Ab10433 RRID:AB-2066656), Parkin (1/1000, Santa Cruz Biotechnology #sc-32282, RRID:AB_628104), Rab7 (1/800, Santa Cruz Biotechnology #sc-376362, RRID:AB-10987863), and Bax (1/1000, Abcam #ab7977, RRID:AB-306191). β-actin (1/10000, Sigma-Aldrich #A1978, RRID:AB-476692) was used as a loading control. Secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse IgG (1/3000, Jackson ImmunoResearch #115-001-003, RRID: AB-2338443) or goat anti-rabbit IgG (1/3000, Jackson ImmunoResearch #111-001-003, RRID: AB-2337910). Image scans of Western blots were used for semi-quantitative analysis. Western blot analysis required 24 cochleae per age and genotype (Table S1). Each experiment with a pool of 8 cochleae was performed in biological and technical triplicate. All results were normalized by β-actin expression.

The renal cortex and collected podocyte were lysed using a lysis buffer on ice for 30 min. Protein was extracted from lysed tissues. The extracted protein was added to 10% SDS-PAGE and separated through electrophoresis. Protein was then transferred from SDS-PAGE to polyvinylidene difluoride membranes. After that, the membrane was moved to 5% nonfat dry milk in PBS + 0.05% Tween 20 and the blocked process was lasted for 1 h. The primary antibodies were then added to membranes and incubated at 4°C overnight. The membranes was washed by PBS and incubated with peroxidase secondary antibody for 1 h at room temperature. Antibodies and dilutions included the following: anti-nephrin antibody (Abcam, UK, Ab136894, 1 : 2000), anti-NOX-4 antibody (Abcam, UK, Ab109225, 1 : 1000), anti-p38 antibody (Abcam, UK, Ab31828, 1 : 1000), anti-p38 (phospho Y182) antibody (Abcam, UK, Ab47363, 1 : 1000), anti-Bax antibody (Abcam, UK, Ab7977, 1 : 500), anti-Bcl-2 antibody (Abcam, UK, Ab7973, 1 : 1000), and anti-GAPDH antibody (Proteintech, Chicago, IL, USA, 10494-1-AP, 1 : 1000). The blots were visualized with LumiGLO reagent and peroxide, followed by exposure to X-ray film. Western blot analyses were performed at least in triplicate.