Anti tubulin antibody

Cells of the HepG2 and HEK293T cell lines were maintained in Dulbecco’s Modified Eagle’s Medium containing 10% inactivated fetal bovine serum. All cell lines were maintained in penicillin (100 IU/mL) and streptomycin (100 mg/mL) in 5% CO₂ at 37°C. The expression constructs of TRIM14 and STAT1 were generated by cloning the sequence of the coding region into a VR1012 expression vector. Site-directed mutagenesis of STAT1 was generated by QuikChange PCR (TransGen, Beijing, China). The pHBV1.3 plasmids were provided by Dr. Lishan Su, University of North Carolina. Human interleukin (IL)-27 recombinant protein was obtained from R&D Systems (Minneapolis, MN, USA). Human IFN-α was purchased from Peprotech (Jiangsu, China). STAT1, p-STAT1(Tyr701), STAT3, and p-STAT3(Tyr705) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). HBsAg antibody was obtained from Thermo (Shanghai, China), HBc antibody was purchased from Abcam (Shanghai, China), anti-tubulin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-GAPDH antibody was obtained from Proteintech. Antibodies against TRIM14 and IFNAR1 were purchased from Abcam (Shanghai, China).

STS was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and the contents were ≥98% by high-performance liquid chromatography. Chloroquine and compound C were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Anti-p62 antibody, anti-AMP kinase (AMPK) antibody, anti-phosphorylated AMPK antibody (Thr172), anti-P70S6K antibody, anti-phosphorylated P70S6K antibody (Thr309), anti-mTOR antibody, anti-phosphorylated mTOR antibody (Ser2448), and anti-myoglobin antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). ATP bioluminescent assay kit was purchased from TOYO Inc. (Tokyo, Japan). Anti-atrial natriuretic peptide (ANP) antibody, anti-tubulin antibody, and fluorescein-conjugated goat anti-rabbit and fluorescein-conjugated goat anti-mouse were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-active caspase-3 antibody was purchased from Abcam Plc. (Cambridge, MA, USA). RNAspin Mini Kit used for isolating total RNA was purchased from GE Healthcare (Buckinghamshire, UK). All other chemicals used were of the highest grade available commercially.

VD3, 25VD, and 1,25VD were purchased from Merck Life Sciences (Milan, Italy) and dissolved in ethanol. Protein kinase C (PKC) inhibitor (GÖ6850) and c-Jun N-terminal kinase (JNK) inhibitor (SP600125) were from Cayman Chemical (Ann Arbor, MI, USA). N-Acetyl-L-Cysteine (NAC), MitoTracker Red CMXRos, and CellROX R Deep Red Reagent were from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). JC-1 was from Adipogen Life Sciences (Liestal, Switzerland). Anti-LC3B antibody was from Proteintech (Rosemont, IL, USA), antitubulin antibody was from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-VDAC and antiphospho-(Ser) PKC substrates antibodies were from Cell Signaling Technology (Danvers, MA, USA). MiR05 was purchased from Oroboros Instruments (Innsbruck, Austria). All other reagents, unless otherwise stated, were from Merck Life Sciences (Milan, Italy).

The HIV-1 proviral DNA clone HIV-1_NL4–3 was obtained from the NIH AIDS Research and Reference Reagent Program. The NLENY1-IRES DNA is a derivative of HIV-1_NL4–3 [40 (link)]. The NLENY1-IRES-ES DNA is similar to NLENY1-IRES except for insertion of stop codon at the beginning of env gene and is therefore Env-negative [40 (link)]. Rab5-GFP was a gift from Stephen Ferguson and Michel Tremblay. The Tet-IFITM1 DNA construct has the cDNA of human ifitm1 gene inserted into the pRetroX-Tight-Pur vector. The IFITM1 mutants Δ(117–125), Δ(112–125) and Δ(108–125), which have different lengths of the C-terminal sequence (illustrated in Fig. 1), have been previously reported [10 (link)]. The ER retention signal KDEL was added to the C-terminus of IFITM1 by a PCR-based method. The cDNA clone of mouse ifitm1 was purchased from ATCC, amplified by PCR, and cloned into pRetroX-Tight-Pur to generate Tet-mIFTM1. Like all human IFITM1 constructs, a Flag tag was added to the N-terminus of mIFITM1 to facilitate detection by western blotting. All these DNA clones were verified by sequencing. The anti-Flag antibody was purchased from Sigma, anti-tubulin antibody from Santa Cruz Biotechnology, FITC-conjugated anti-HIV-1 p24 antibody from Beckman, anti-calreticulin antibody from Abcam, and BMQC from Invitrogen. The HEK293 cells and SupT1 cells were originally obtained from ATCC.

Total cellular protein for HT29 cells and control cells were collected using a lysis mix in mammalian cell lysis solution–CelLytic M (Sigma-Aldrich Pte Ltd, USA) in accordance with manufacturer’s instructions. Equal protein concentration (20 µg) from each samples were added onto 10% Mini-PROTEAN TGX (Bio-Rad Laboratories, CA, USA) and separated by electrophoresis. Separated proteins were transferred onto nitrocellulose membrane (Bio-Rad Laboratories, CA, USA). The membranes were incubated with mouse anti EV71 antibody (AbD serotech, Oxford, UK) or anti tubulin antibody (Santa Cruz Biotechnology inc, California, USA) respectively in shaker 4°C overnight. The membranes were washed three times in Tris-buffered saline with 0.05% tween-20 before and incubating with secondary antibodies conjugated to horseradish peroxidase (HRP) for 30m. (ThermoScientific, Waltham, USA). After three wash with Tris-buffered saline and 0.05% tween-20, membrane was subjected to Clarity Western ECL Substrate (Bio-Rad Laboratories, CA, USA) in accordance with manufacturer’s instructions. The membranes were then scanned using the LI-COR C-DiGit Blot Scanner (LI-COR Biosciences, Lincoln, Nebraska, USA).