Anti β actin antibody sc 47778

Cells (2 × 10⁵) were plated in 60 mm dishes, treated with the indicated concentrations of rapamycin for 0~48 h, and then lysed using RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], and 0.5% sodium deoxycholate) containing protease and phosphatase inhibitors (Sigma-Aldrich, San Diego, CA, USA). Proteins were quantified using a Bradford Protein Assay kit (Pierce Biotechnology, Rockford, IL, USA). For Western blot assay, 20 μg of proteins was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (GE HealthCare, Hatfield, UK), which were then treated with anti-PROX1 antibody [25 (link)], anti-phospho-MTOR (p-MTOR, ser-2448) antibody (sc-101738, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MTOR antibody (Ab-2448, Cusabio, Houston, TX, USA), anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, CA), appropriate secondary antibodies, and enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

DMN was purchased from Wako Pure Chemical Industries (147–03781, Richmond, VA, USA). Recombinant mouse GM-CSF was obtained from Chemicon (Temecula, CA, USA). Anti-collagen type I antibody (ab34710, rabbit polyclonal), anti-α-SMA antibody (ab5694, rabbit polyclonal), and goat anti-rabbit IgG H&L (HRP) antibody (ab205718) were purchased from Abcam (Cambridge, MA, USA). Anti-β-actin antibody (sc-47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-TGF-β1 antibody was purchased from Sigma (St. Louis, MO, USA); and anti-peroxisome proliferator-activated receptor-gamma (PPAR-γ) antibody (A3409A) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

XL1 Blue cells were from Agilent
Technologies (Santa Clara, CA, USA). BL21 (DE3) pLysS chemical competent
cells were from Invitrogen. pET-15b and pET-28a plasmids for protein
expression were from Novagen (Madison, WI, USA). pET plasmids were
constructed for protein expression, including pET28a-Uba1, pET15b-UbcH7,
pET15b-UbcH5b, pET28a-E6AP, pGEX-4T-1-Parkin, pGEX-4T-1-HHARI, pET15b-UB,
and pET28a-M^pro. PINK1 was purchased from Boston Biochem
(AP-182–100). HEK293T cells were from American Tissue Culture
Collection (ATCC) and cultured in high-glucose Dulbecco’s modified
Eagles medium (DMEM) (Life Technologies, Carlsbad, CA, USA) with 10%
(v/v) fetal bovine serum (FBS) (Life Technologies). The anti-UB antibody
(sc-8017), anti-Myc antibody (sc-40), anti-HA antibody (sc-7392),
anti-GFP antibody (sc-9996), anti-E6AP antibody (sc-25509), anti-Parkin
antibody (sc-32282), and anti-β-actin antibody (sc-47778) were
from Santa Cruz Biotechnology. The anti-Flag M2 antibody (F3165) was
from Sigma-Aldrich. The anti-Mfn1 antibody (A9880), anti-Mfn2 antibody
(A12771), and Rabbit anti-GFP antibody (AE078) were from ABclonal
Technology. The antibodies were diluted between 500- and 1000-fold
to probe the Western blots.

Total protein extracts from rat liver by MRJP1 or casein treatment for 7 days by the same procedures as described in Expt. 1. 200 mg of liver tissue was homogenized in 4 ml lysis buffer containing Protease Inhibitor Cocktail as previously described [26] (link) with a slight modification. CYP7A1 or β-actin was detected using an anti-CYP7A1 antibody (ab65596, Cambridge, UK) or anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology) respectively, followed by incubation with a peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) respectively. Proteins were detected using an ImmunoStar LD Western blotting detection system (Wako Pure Chemical). After detection, NIH-Image J was used for band quantification.

Sinapic acid (SA, 3,5-dimethoxy-4-hydroxycinnamic acid), LPS, and ATP were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nigericin was purchased from Tocris (Bristol, UK). Poly (dA:dT) and Salmonellatyphimurium flagellin were purchased from InvivoGen (San Diego, CA, USA). Anti-IL-1β antibody (AF-401-NA) was purchased from R&D Systems (Minneapolis, MN, USA). The anti-caspase-1 (AG-20B-0042), anti-NLRP3 (AG-20B-0014), and anti-ASC (AG-25B-0006) antibodies were purchased from AdipoGen Life Science (San Diego, CA, USA). Anti-IL-6 (12912), anti-phospho-STAT3 (9145), anti-phospho-IκBα (9246), anti-phospho-JNK (4668), and anti-phospho-ERK (9106) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-β-actin antibody (sc-47778) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). All culture reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Relative expression levels of hCLOCK, RhoA, Rac1, IL-6, ROCK1, COX2, Phospho-NF-κB p65, and β-actin were measured by Western blotting using standard methods. Anti-Rac1 (ab15880) and anti-hCLOCK (ab98948) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-IL-6 antibody (21865-1-AP) was purchased from Proteintech (Chicago, IL, USA). Anti-ROCK1 (# 4035), anti-COX2 (# 4842), and anti-Phospho-NF-κB p65 (Ser536, # 3033) antibodies were purchased from Cellsignal (Beverly, MA, USA). Anti-β-actin antibody (sc-47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Band intensities of RhoA were normalized to the band intensities of the total RhoA. The band intensities of the other proteins were normalized to the band intensity of the cell structural protein β-actin.