Beta actin ab8227

HSCs were treated with 27 μM nortriptyline, 75 μM of B13, 25 μM of ceramide-C6, or ethanol vehicle for 48 hours and then washed in ice-cold dPBS and harvested in 100 ul of RIPA Buffer (Boston BioProducts) containing protease and phosphatase inhibitors (Sigma). Cellular lysates (30 mg) were prepared in Laemmli’s sample buffer (Boston BioProducts), separated by electrophoresis on a 12% polyacrylamide gel, and transferred to a polyvinylidene difluoride membrane (Millipore). Membranes were blocked with Tris-buffered saline 0.05% Tween 20 (TBS/T) containing 5% non-fat dry milk. Membranes were incubated with primary antibodies overnight at 4 °C and with secondary antibodies conjugated to horseradish peroxidase (HRP) (Cell Signaling Technology) for 1 hour at room temperature. Membranes were then washed three times with TBS/T. Immunoreactive bands were visualized on a C-Digit blot scanner (Li-Cor Biosciences) with a chemiluminescent HRP substrate (GE Healthcare). The following antibodies were used: αSMA (ab5694) and beta actin (ab8227) (Abcam).

Western blotting was carried out as described previously [5 (link)].
Antibody dilutions: Carbonic anhydrase II ab6621 (Abcam) 1:7000 dilution in 3% (w/v) BSA in TBS-T; NADH dehydrogenase flavoprotein 2 ARP57510-PO50 (Cambridge Bioscience) 1:5000 dilution in 3% (w/v) BSA in TBS-T; Beta-actin ab8227 (Abcam) 1:5000 dilution in 3% (w/v) BSA in TBS-T; Carbonic anhydrase III AP7633a (ABGENT) 1:2500 dilution in 3% (w/v) BSA in TBS-T, GAPDH G9545 (SIGMA) 1:5000 dilution in 3% (w/v) BSA in TBS-T and COXIV (ab16056) 1:5000 dilution in 3% (w/v) BSA in TBS-T. Brain mitochondrial samples were normalised to beta-actin level. The average of four samples for each condition (old and young) were plotted showing the mean +/− SEM. The muscle mitochondrial samples were normalised to GAPDH level. The average of the four samples for each condition (old and young) were plotted showing the mean +/− SEM. The retina mitochondrial samples were normalised to COXIV level. The average of the six samples for each condition (young and old) were plotted showing the mean +/− SEM. Statistical analyses (unpaired t-tests with Welch's correction) were carried out in GraphPad Prism.

Antibodies were purchased from the following companies: phosphor-S6K (Thr 389), S6K, mTORC1, pACC, ACC, caspase 3, cleaved PARP, TSC1, and TSC2 were purchased from Cell Signaling Technology, Danvers, MA, USA; beta-actin (ab8227) from Abcam, Cambridge, UK; beta-actin (937215) from R&D systems, Minneapolis, MN, USA; CD63 from Invitrogen, Waltham, MA, USA; CD81 from Santacruz; and goat anti-mouse IgG (H + L) secondary antibody (HRP), and goat anti-rabbit IgG (H + L) secondary antibody (HRP) from Thermo Fisher Scientific, Waltham, MA, USA. Reagents were obtained from the following sources: DAPI was obtained from Thermo Fisher Scientific; goat anti-mouse Alexa Fluor 488, goat anti-mouse Alexa Fluor 594, goat anti-rabbit Alexa Fluor 488, and goat anti-rabbit Alexa Fluor 594 were obtained from Invitrogen; phosphatase inhibitors, protease inhibitors, calpeptin, GW4869, and DMSO were obtained from Sigma-Aldrich, St. Louis, MO, USA; TSC1 and TSC2 siRNAs from Invitrogen; CCK-8 from Dojindo; rapamycin from Merck, Darmstadt, Germany; Lipofectamine 2000 and Exosome isolation buffer kit from Thermo Fisher Scientific; and glucose-free DMEM and high-glucose DMEM from Welgene, Gyeongsan, Korea.

Forskolin was purchased from Sigma (USA). PCI-34051 and Tubastatin were procured from Cayman chemicals. DMEM, FBS, 1% Penicillin & Streptomycin Antibiotics were obtained from Himedia. Monoclonal antibody of HDAC8 (A-4008) was obtained from Epigenetik, Beta actin ab8227 (Abcam), GAPDH (MA5–15738), Alpha tubulin (B-5-1-2) and anti-acetylated alpha tubulin (Cat no: 322700) were purchased from Thermo scientific Life technologies respectively. Protein A Agarose beads were obtained from Santa Cruz. Poly-L-Lysine (Sigma P8920), 4′, 6-Diamindino-2-phenylindole dichloride DAPI as a nuclear stain (Sigma), Alexa Fluor® 555 Dye (Thermo Fischer Scientific). HDAC8 FLUOR DE LYS fluorometric assay kit (BML-AK518–0001) was purchased from Enzo life sciences. Propidium iodide was purchased from Himedia. GST Column (GE Healthcare 17–5132-01), Custom synthesized peptides of alpha tubulin 33–46 amino acids, Acetylated alpha tubulin peptide, at Lys40 DGQMPSDKTIGGGD and Unacetylated Alpha tubulin peptide DGQMPSDKTIGGGD from SIGMA (USA) (resuspended in MilliQ water).

Protein lysates were prepared using RIPA buffer containing 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, and 30 μL/mL aprotinin. The proteins (20–30 μg) were resolved in SDS-PAGE, electro-transferred onto nitrocellulose membranes (Hybond-ECL, GE Healthcare) and blocked with 5% BSA. Primary antibodies (1:1000 dilution) included NRF2 (D1Z9C), BiP/GRP78 (C50B12) and CHOP (L63F7) from Cell Signaling; beta-actin (ab8227) from Abcam and ATF3 (C19) from Santa Cruz. After secondary antibody incubation (1:3000, 2 h), the proteins were detected using Lumiglo substrate (Cell Signaling Technology, CA).

Granulosa cells were collected in Laemmli buffer (Bio-rad) containing DTT (Omnipur), phosphatase and protease inhibitors (G Biosciences), and were boiled at 95°C for five minutes. Protein extracts were resolved by polyacrylamide electrophoresis and transferred to nitrocellulose membranes. After blocking with 5% milk in TBS-T, the membranes were incubated overnight at 4°C with primary antibodies (1:1000) followed by washing with TBS-T (3X 10 min each) and incubation with secondary antibody (1:10000) for 1hour at room temperature. The immunoblotted proteins were detected using Immun-Star Kit and Chemidoc Analyzer (BioRad). Whenever necessary, the membranes blotted with one primary antibody were stripped using stripping buffer (10% SDS, 0.5 M Tris-Hcl, DEPC H2O ml and 2-mercaptoethanol) and re-blotted with another antibody. Antibodies used: MAPK3/1 (#4695), phoshpho-MAPK3/1 (Thr202/Tyr204)(#4376) from Cell Signaling; EGR-1 (#sc-189x) form Santa cruz biotechnology; beta actin (ab8227) and Goat anti-rabbit-IgG (ab6721) from Abcam.