Anti mouse igg a4416

Whole cell extracts of cultured cells were prepared in radioimmunoprecipitation (RIPA) lysis buffer supplemented with phosphatase and protease inhibitors (Calbiochem, Burlington, MA, USA). Protein concentration was determined by the bicinchoninic acid assay (BCA) (Thermo Fisher Scientific, Waltham, MA, USA) and separated by SDS-PAGE gel. The following antibodies and dilutions were used: hnRNPA1 (1:2000, Cell Signaling, Danvers, MA, USA), cleaved PARP1 (1:1000, Cell Signaling), HSP90 (1:4000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), GAPDH (1:3000, EMD Millipore, Billerica, MA, USA), Survivin (1:2000, Cell Signaling), and Flag-tag (1:2000, Sigma-Aldrich). Following blocking with 5% BSA, membranes were incubated with the primary antibodies overnight at 4 °C. The next day, membrane was incubated with secondary antibodies for 1 h at room temperature. Secondary anti-mouse IgG (A4416) and anti-rabbit IgG (A6667) antibodies were purchased from Sigma-Aldrich and used at a 1:4000 dilution. Images of blots were acquired on HyBlot ES Autoradiography Film (Thomas Scientific, Swedesboro, NJ, USA) following incubation with SuperSignal West Pico PLUS (Thermo Fisher Scientific). When necessary, membranes were stripped using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) according to manufacturer’s instructions.

Cells treated with RA and controls were lysed with 1× lysis buffer (CellLysis Buffer, Cell Signaling Technology, Danvers, MA, USA), and protein was quantified using the Bradford protein assay (Thermo Fisher Scientific). For blotting, 20 μg of protein were separated by SDS-PAGE and transferred to a PVDF membrane. After 1 h under blocking solution (5% Milk in TTBS), the membrane was incubated overnight at 4 °C with primary antibodies against SOX2 (ab97959, 1:2000 dilution; Abcam, Cambridge, UK), p21 (sc-6246, 1:500 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), and β-actin (ACTB) (sc-47778, 1:2000 dilution; Santa Cruz Biotechnology) as protein control. Incubation of primary antibodies was followed by incubation with secondary antibodies (1:10,000 dilution; anti-rabbit IgG 7074, Cell Signaling; or anti-mouse IgG a4416, Sigma-Aldrich) for 1 h. Chemiluminescence was detected using ECL Western Blotting Substrate (Thermo Fisher Scientific) and analyzed using iBright (Thermo Fisher Scientific). The immunodetection signal was analyzed using ImageJ.

Primary antibodies were purchased and used at a dilution of 1:500 (unless indicated otherwise) as follows: HAVcR-1 Pab13202, (Abnova, Heyford, Oxon, UK); TIM-1/HAVcR-1 AF1817, E-cadherin 17029 (R&D Systems, Abingdon, Oxon, UK); TIM-1 (N-13 HAVcR-1) SC47495, GAPDH SC32233, Claudin-1 SC17658, Claudin-7 SC177670, Occludin SC8145, ZO-1 SC8146, PKM2 SC65176, cyclin D1 SC753 (Insight Biotechnology LTD, Middlesex, UK); α-catenin C1620 (BD Transduction Laboratories, San Jose, CA, USA); β-catenin 8415 (Sigma-Aldrich, Gillingham, Dorset, UK); Eplin A300-103A (Bethyl Labs, Montgomery, TX, USA). Secondary antibodies were prepared as per manufacturer’s instructions and were purchased as follows: Anti-Mouse IgG A4416, anti-Rabbit IgG A6154, anti-Goat IgG A5420 (Sigma-Aldrich, Gillingham, Dorset, UK); Biotinylated anti-Mouse IgG (Vector Labs, Orton Southgate, Peterborough, UK); AlexaFluor 488 anti-Rabbit, anti-Mouse, anti-Goat and AlexaFluor 594 anti-Rabbit, anti-Goat, DAPI (Thermo Fisher Scientific, Cramlington, UK).