Actin mab1501

Monoclonal antibodies recognizing HCV core (ab2740) and NS3 (ab65407) were purchased from Abcam (Cambridge, UK); ApoJ (ARG62961) for IFA from Arigo Biolaboratories (Taipei, Taiwan); actin (MAB1501) from Millipore (Billerica, MA); and DsRed (tcba13674) from Taiclone Biotech Corp. (Taipei, Taiwan). Polyclonal antibodies recognizing human ApoJ for western blot (WB) analysis (ab69644) was purchased from Abcam; human ApoJ (sc-6419) for immunoprecipitation from Santa Cruz Biotechnology (Santa Cruz, CA); mouse ApoJ for WB analysis (PA5-46931) from Thermo Fisher Scientific Inc. (Waltham, MA); SOAT1 (ARG56476) and SOAT2 (ARG57814) for WB analysis from Arigo Biolaboratories; and SOAT 1 (bs-7544R) and SOAT 2 (bs-5020R) for IFA from Bioss Antibodies (Beijing, China). Goat anti-mouse Alexa-488-, and anti-rabbit Alexa-568-conjugated secondary antibodies were purchased from Thermo Fisher Scientific Inc.; Goat anti-mouse HRP- and anti-rabbit HRP-conjugated secondary antibodies were purchased from Chamot Biotechnology Co. Ltd (Shanghai, China).

For immunoblot assay, cells were first subjected to lysis in NuPAGE LDS Sample Buffer. Total proteins content from every samples was measure using Pierce BCA Protein Assay Kit according to the manufacturer’s recommendations. Samples lysates were then loaded onto NuPAGE 4–12% Bis-Tris Protein Gels (10, 12, or 15 wells). After electrophoresis, proteins were transferred to nitrocellulose membranes. The transblotted membranes were blocked for 1 h and then probed with appropriate primary antibodies (dilution as recommended by manufacturers) overnight at 4 °C. Next, the membranes were washed three times for a total of 30 min and then incubated with secondary antibodies at room temperature for 1 hr. After another three washes, proteins were detected using SuperSignal West Pico PLUS chemiluminescent Substrate, PXi imager (Syngene), and GeneSys software (Syngene, Paris, France), according to the manufacturer’s manual. Proteins expression were quantified using GeneTool software (Syngene) and normalized to their corresponding loading control. Antibodies for immunoblotting were purchased from the following sources: FASN (#3180S) from Cell Signaling Technology; Actin (MAB1501) from Millipore; IDH1 R132H (DIA-H09) from Dianova. HRP conjugate anti-rabbit (W4011) and anti-mouse (W4021) secondary antibodies were purchased from Promega (Charbonnières les Bains, France).

Tissues or cells were lysed in ice-cold lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EGTA, 1% NP-40, 0.25% sodium deoxycholate, 1 mM sodium pyrophosphate 1 mM sodium vanadate, 1 mM NaF, 10 mM sodium β-glycerolphosphate, 0.2 mM PMSF and a 1:1000 dilution of protease inhibitor cocktail (Sigma-Aldrich, Saint-Louis, MI, USA). Protein concentration was determined using a BCA assay (Pierce, Rockford, IL, USA). Equal amounts of protein were resolved by LDS-PAGE (4–12% gel; NuPAGE, Invitrogen, Basel, Switzerland) and electro-transferred onto nitrocellulose membranes (0.2 μm, BioRad, Reinach, Switzerland). Equal protein loading on membranes was checked by Ponceau S staining. Blots were blocked in tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl) containing 0.1% Tween (TBS-T) supplemented with 5% non-fat dry milk. Membranes were then placed in a 50 ml Falcon tube and incubated overnight at 4°C with gentle rotation with the respective primary antibody solutions. Antibody-antigen complexes were detected by using the ECL system and detected with the Fuji LAS-3000 image reader (Fujifilm, Tokyo, Japan). The following primary antibodies (diluted 1:1000) were used Actin, MAB1501 (Millipore, Darmstadt, Germany); p53, #2524, p38, #9212 and phospho-p38, #9211, HSP90, #4877 (Cell Signalling, Danvers, MA, USA), p21, ab109199 (Abcam, Cambridge, UK).

Antibodies used throughout the paper include Matrin 3 ab151714 and ab70336 (abcam) and HPA036565 (Sigma), Flag F3165 (Sigma) and 2368 (Cell Signaling), actin MAB1501 (Millipore), Aly ab6141 and ab202894 (abcam), ddx39b 14798–1-AP (Proteintech) and NBP2–52456 (Novus Biologicals), Sarnp HPA030902 (Sigma), and GAPDH 2118 S (Cell Signaling).

The DK from E. cava was prepared in previous our studies [20 (link),23 (link),25 (link)]. The MTT was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The Z-VAD-fmk, 3MA, HCQ, E64D, and pepstatin A were purchased from Selleck Chemicals (Houston, TX, USA). The primary antibodies against caspase-3 (#2696), cleaved caspase-3 (#9664), PARP (#9542), cleaved PARP (#5625), Bcl-2 (#4223), Bax (#5023), Bim (#2933), Bak (#12105), LC3B (#3868) were purchased from Cell Signaling Technology (Danvers, MA, USA). Sequestosome 1 (SQSTM1, p62, sc-28359) and LAMP-1, (sc-20011) were purchased from Santa Cruz (St. Dallas, TX, USA), and actin (MAB1501) was purchased from Millipore (Billerica, MA, USA). All the remaining chemicals and reagents were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany).