Actin antibody

Frozen liver and EWAT were homogenized on ice in RIPA Buffer (Sigma Chemical, Inc.) (10:1 buffer to QUAD mass and 5:1 buffer to EWAT mass ratios) containing protease and phosphatase inhibitor cocktails (Halt Protease/Halt Phosphatase, Thermo Fisher). Homogenates were rotated at 4°C for 1 h and centrifuged at 9,000 x g for 30 min at 4°C. The protein concentrations of the supernatants were determined by the BCA method (Pierce, Inc), and equal amounts of protein were subjected to SDS-PAGE along with molecular weight standards (Bio-Rad, Hercules, CA and Magic Mark, Invitrogen). Proteins were then electrophoretically transferred to Immobilon membranes and immunoblotted with Cidea and Cidec antibodies. Light generated by the alkaline phosphatase conjugated secondary antibody and CDP-Star reagent was detected using a digital imaging system (UVP, Upland, CA). To account for gel loading differences, all immunoblots were stripped and re-probed with a ß-tubulin antibody. Relative signal intensities of immunoreactive bands were determined using Total Lab software (Nonlinear, Inc., Durham, NC) or GraphPad Prism and ImageJ. All data were normalized to ß-tubulin and expressed as a percentage of LFD-Vehicle. The ß-actin antibody was from Cell Signaling, the Cidea antibody was from Sigma, and the Cidec antibody was a kind gift from Cynthia Smas, Toledo, Ohio.

All cell lines were obtained from ATCC except SUM149 and SUM159 cells were obtained from the lab of Dr. Charles Perou, and SUM229 parental and EpCAM-sorted cells were obtained from the lab of Dr. Gary Johnson. SUM149, SUM159, SUM229, and EpCAM-sorted SUM149 and SUM229 cells were cultured in HuMEC medium (Gibco, Waltham, MA, USA) with supplements added and 5% FBS. MDA-MB-231 cells were cultured in DMEM (Gibco) with 10% FBS. All other cell lines were cultured in RPMI-1640 (Gibco) with 10% FBS. Penicillin/Streptomycin (Gibco) was added to all culture media. Cells were occasionally tested for mycoplasma with the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland). DRD2 antibody was obtained from Millipore (AB5084P) and used at 1:1000. Actin antibody was obtained from Cell Signaling Technologies. Thioridazine and quinpirole were obtained from Sigma-Aldrich, and SKF83959 was obtained from Cayman Chemical.

Protein from treated and untreated cell lysates was estimated using the BCA protein estimation assay (Thermo Scientific). For Western blotting, anti-HSP70 antibody (Enzo life sciences), and anti-Sp1 antibodies (Cell Signaling) were used to check for levels of different proteins in the lysates. Actin was used as loading control. Actin antibody was obtained from Cell Signaling Technology.

Urolithin A was purchased from Cayman Chemical Company (#22,607). Bafilomycin-A1 (#B1793) and anti-p62/SQSTM1 antibody (#P0067) were purchased from Sigma-Aldrich. Anti-amyloid beta peptide antibody (MOAB-2) was purchased from Millipore (#MABN254). AT8 phospho-Tau antibody (Ser202, Thr205) was purchased from Thermo Fisher Scientific (#MN1020). Anti-β-amyloid antibody (6E10) (#803,017) was purchased from Biolegend. LC3 antibody was purchased from Proteintech (#14,600–1-AP). Actin antibody was purchased from Cell Signaling Technology (#3700) goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor™ 488, was purchased from Sigma-Aldrich (#A-11001). DMEM/F12 (#11,330,032) and DQ-BSA red (#D12051) were purchased from Thermo Fisher Scientific. Earl’s balanced salt solution (EBSS) (#24,010–043) was purchased from Gibco. DMEM (#10–013-CV), fetal bovine serum (#35–010-CV) and penicillin–streptomycin solution (#30–002-CI) were purchased from Corning. Beta amyloid oligomers were generated according to the manufacturer’s instructions using the beta amyloid (1–42), aggregation kit (#A-1170–2) purchased from rPeptide.

HEK293T cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). ACHN and 786-O cells were provided by Shanghai GK Genentech Co., Ltd. (Shanghai, China). Cells freshly amplified and frozen were used every 2–3 months. They were routinely tested for the absence of mycoplasma contamination. Dulbecco’s modified Eagles medium (DMEM) high glucose medium and RPMI 1640 medium were purchased from Hyclone (Thermo Fisher Scientific, Inc.). Fetal bovine serum was purchased from Gibco (Thermo Fisher Scientific, Inc.). Transfection reagent Lipofiter™ was purchased from HanBio (Shanghai, China). TRIzol was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). A complementary DNA (cDNA) kit was purchased from Fermentas (Thermo Fisher Scientific, Inc.), and a SYBR Green real-time polymerase chain reaction (PCR) kit was purchased from Applied Biosystems (Thermo Fisher Scientific, Inc.). ABAT and ALDH6A1 antibodies were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). ACTIN antibody and Flag-tag antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). A water-soluble tetrazolium salt (WST)-1 cell proliferation assay kit was purchased from Roche (Mannheim, Germany). Horseradish peroxidase (HRP)-tagged secondary antibody and ECL kits were purchased from Pierce (Thermo Fisher Scientific, Inc.).