Mouse monoclonal anti tubulin antibody

According to the manufacturer’s instructions, proteins were extracted from cells or tissues using Western-IP Lysis Buffer (Beyotime, Shanghai, China). The protein concentration was determined using a bicinchoninic acid (BCA) protein concentration determination assay kit (Beyotime, Shanghai, China). Prepared samples were electrophoresed in a 10% SDS-PAGE gel and blotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) using the Tetra Handcast system (Bio-Rad, USA). The membranes were blocked for 3 h at room temperature and incubated overnight at 4°C with an appropriate primary antibody in Tris-buffered saline with 0.05% Tween (TBST) containing 5% non-fat milk. After washing in TBST, the membranes were incubated with secondary antibody, washed and visualized using a supersensitive enhanced chemiluminescence (ECL) kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s protocol. The protein bands were detected and quantified using the Gene Gnome Syngene Bio Imaging System (SYNGENE, UK).
Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).

Rabbit polyclonal anti-myc antibody, mouse monoclonal anti-actinin-1 antibody, and DMSO were from Sigma–Aldrich (St Louis, MO, USA). Mouse monoclonal anti-Stx6 antibody was from BD Transduction Laboratories (San Jose, CA, USA). Rabbit polyclonal anti-Stx6 and anti-Stx16 antibodies were from Synaptic Systems (Goettingen, Germany). Mouse monoclonal anti-Tubulin antibody was from Abcam (Cambridge, MA, USA). Human holo-transferrin conjugated to A488 was from Invitrogen (Grand Island, NY, USA). Mouse anti-myc (c-Myc 9E10) and rabbit anti-furin (H-220) were from Santa Cruz Biotechnology (Dallas, TX, USA). Polyclonal anti-P-Akt(308) and P-Akt(473) were obtained from Cell Signaling Technology (Danvers, MA, USA). Cy3- and A488-conjugated donkey anti-rabbit and donkey anti-mouse secondary antibodies and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Nocodazole was purchased from EMD Biosciences Inc. (Darmstadt, Germany) (10 mM stock in DMSO) and C2-ceramide was purchased from Enzo Life Sciences (Farmingdale, NY, USA) (50 mM stock in DMSO). Pre-designed siRNA for Stx6 (siStx6: 5′-CCGAGTCATCAGAAGAACTAA-3′) and non-related (siNR: 5′-AATAAGGCTATGAAGAGATA C-3′) were from Qiagen (Valencia, CA, USA). Human insulin was purchased from Eli Lilly (Indianapolis, IN, USA).