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3 protocols using ab198202

1

Comprehensive Protein Analysis Protocol

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SNRPB (ab85534, Abcam) and RAB26 (ab198202, Abcam) were used for western blot assays. SNRPB (16807–1-AP) and RAB26 (14284–1-AP) were obtained from Proteintech for IHC studies. Vimentin, MMP2, MMP9, ki67, ERK, pERK, and actin were from Abclonal. Flag-M2 antibody was obtained from Sigma. LC3B, AKT, and pAKT antibodies were obtained from CST.
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2

Western Blot Analysis of Protein Expression

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Total proteins from A549 cells and tumor tissues were isolated using RIPA buffer (Beyotime) on ice. Then equal amount of protein samples were separated by 10–12% SDS-PAGE and transferred to PVDF membranes (Millipore), and then blocked with 5% bovine serum albumin (BSA; Beyotime). After being incubated with primary antibodies at 4°C overnight, samples were subjected to goat anti-rabbit HRP-conjugated secondary antibody (ab6721, 1:10,000, Abcam) at room temperature for 2 h. The bands were finally visualized by an ECL kit (Thermo Scientific), and then intensity was quantified by Image J 1.51 software (National Institutes of Health). Primary antibodies used included: RAB26 (ab198202; 1:000; Abcam), matrix metalloproteinases (MMP)2 (ab92536; 1:1000; Abcam), MMP7 (ab216631; 1:1000; Abcam), Bcl-2 (ab182858; 1:2000; Abcam), Bax (ab32503; 1:5000; Abcam), cleaved caspase 3 (#9661; 1:1000; Cell signaling pathway), caspase 3 (#9662; 1:1000; Cell signaling pathway), Bim (ab32158; 1:1000; Abcam), APAF-1 (ab234436; 1:1000; Abcam), Ki67 (ab16667; 1:1000; Abcam), proliferating cell nuclear antigen (PCNA; ab92552; 1:1000; Abcam), SMAD3 (ab208182; 1:1000; Abcam) and GAPDH (ab181602; 1:5000; Abcam).
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3

Cell Line Cultivation and Antibody Validation

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Human NPC cell lines (S26 and 5–8F) were kindly gifted by Professor Chaonan Qian at SYSUCC and human embryonic kidney 293T cells were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences. All cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, NY, USA) and cell cultures were maintained in a 5% CO2 humidified incubator at 37 °C. No evidence of mycoplasma contamination was observed using mycoplasma detection kit (Vazyme, Nanjing, China). Primary antibodies were commercially available, including those for GOLIM4 (PAB28477, Abnova, Taipei, China), β‐actin (A1978, Sigma‐Aldrich, St. Louis, USA) and RBFOX2 (HPA006240, Sigma‐Aldrich, St. Louis, USA, ab57154, Abcam, Cambridge, USA), Tubulin, CCND1, C‐MYC, N‐Cadherin, and Vimentin (2144, 2922, 9402, 14215, 5741, Cell Signaling Technology, Danvers, USA), RAB26 (ab187151, ab198202, Abcam, Cambridge, USA), E4F1 (H00001877‐M03, Abnova, Taipei, China) and HA tag (ab9100, Abcam, Cambridge, USA, H6908, Sigma‐Aldrich, St. Louis, USA).
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