Ab133734

Formalin-fixed and paraffin-embedded (FFPE) glioma tissues were cut into 3-4 μm sections, then deparaffinised using xylene, and hydrated through graded alcohol. Perform heat mediated antigen retrieval with citrate buffer (pH = 6.0) before commencing with IHC staining protocol. In short, the sections were incubated with PTBP1 antibody (ab133734, Abcam, USA) overnight at 4°C. Then, the secondary antibody (Dako, Denmark) was applied and incubated for 1 h at room temperature, followed by 3,3-diaminobenzidine tetra hydrochloride staining, and observed under microscope (BX51, Olympus, Japan). The expression of PTBP1 was calculated as the sum of the percent positivity of stained tumor cells and the staining intensity. The percent positivity was scored as follows: 1 for 0-25%, 2 for 26%-50%, 3 for 51%-75%, and 4 for >75%. The staining intensity was scored as follows: 0 for no staining, 1 for light yellow, 2 for yellowish brown, and 3 for dark brown. A final staining score of ≥6 was defined as high expression. All the samples were scored separately by three independent pathologists, who were blinded to the clinical data.

Protein samples were prepared using RIPA lysate (Thermo Fisher Scientific, Waltham, MA, USA), resolved via sodium dodecyl sulfonate-polyacrylamide gel electrophoresis, and then transferred to PVDF membrane (Millipore, Billerica, MA, USA). Next, membranes were incubated with primary antibody (including antibody against Bax [ab32503; 1:1,000; Abcam, Cambridge, UK], Bcl2 [ab32124; 1:1,000; Abcam], glucose transporter protein 1 [GLUT1, ab115730; 1:1,000; Abcam], lactate dehydrogenase A [LDHA, ab52488; 1:1,000; Abcam], and PTBP1 [ab133734; 1:1,000; Abcam]) followed by incubation with HRP-tagged secondary antibody (Abcam). Anti-β-actin antibody was used as a control. The blots were detected by an ECL kit (Beyotime)

Total protein lysates were extracted from tissue culture cells using Laemmli buffer, boiled and the protein concentration determined (BCA protein assay, Pierce). Thirty micrograms of protein was resolved on SDS-PAGE gels, transferred to PVDF membranes and detected with a rabbit monoclonal antibody for NOVA1 (Abcam, EPR13847, ab183024, 1:1000 dilution in 5% NFDM), PTBP1 (Abcam, EPR9048B, ab133734, 1:10,000 dilution in 5% NFDM), or PTBP2 (Abcam, EPR9891, ab154853, 1:1000 dilution in 5% NFDM). Protein loading was determined with antibodies against with histone H3 (Anti-Histone H3 antibody produced in rabbit, H0164; Sigma). Blots were imaged with Bio-Rad Chemidoc XRS + Molecular Imager and quantified with Bio-Rad Image Lab software. Analysis shown in Fig. 2b was one cell lysate with the blot repeated twice. Blots shown in Fig. 5b were repeated twice from two separate pulldowns. Figure 5d–f were performed twice in the laboratory with three biological replicates. Figure 6a and b blots were from 1 biological replicated repeated twice in the laboratory. Figure 6c–h are from biological triplicates repeated twice in the laboratory.

Proteins were separated by SDS-PAGE on 8% or 10% polyacrylamide gels and transferred to 0.45-μm nitrocellulose membranes (Whatman Protran). The membranes were incubated with the appropriate primary and secondary antibodies and washed with TBS-Tween 20. Horseradish-peroxidase-conjugated secondary antibodies were detected by SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific; PI-34080). Antibodies used were anti-pol II p-Ser2 (Abcam; ab5095) (1:500), anti-pol II p-Ser5 (Abcam; ab5408) (1:1000), anti SNRPC (U1C) (Abcam; ab157116) (1:200), anti-U2AF2 (a gift of Prof. Juan Valcarcel, Centre for Genomic Regulation, Barcelona, Spain) (1:500), anti-U2AF35 (Abcam; ab172614) (1:250), anti-FUS (Abcam; ab23439) (1:400), anti-SAP155/SF3B1 (MBL; D221-3) (1:1000), anti-NXF1/TAP (Santa Cruz Biotechnology; sc- 32319) (1:500), anti-GAPDH (GenScript; A00191-40) (1:1000), anti-PTBP1 (Abcam; ab133734) (1:5000), anti a-tubulin (abcam; ab18251) (1:40000), anti-histone 4 (Millipore;05-858) (1:30000), donkey anti-rabbit IgG (Abcam; ab97064), and goat anti-mouse IgG (Abcam; ab7068).

The cells were lysed with radio immunoprecipitation assay (RIPA) peptide lysis buffer (R0010, Beijing Solarbio Science and Technology Co. Ltd., Beijing, China) to extract the total protein. The proteins were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a polyvinylidene fluoride membrane. The membrane was incubated with rat anti-human primary antibodies PTBP1 (ab133734, 1: 1000, Abcam Inc., Cambridge, UK), GLUT3 (ab41525, 1: 1000, Abcam), HK2 (ab209847, 1: 1000, Abcam), and PDK1 (ab207450, 1: 1000, Abcam) at 4° C overnight. Next, the membrane was washed three times with TBST (5 min per time), followed by incubation with horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) (1: 20000; ab205718, Abcam) for 1 h. Protein blots were visualized by ECL-associated fluorography (Merck Millipore, Billerica, MA, USA). The data was analyzed using ImageJ 1.48u (National Institutes of Health Inc., USA). The protein content was reflected by the ratio of gray value between proteins and the internal reference (GAPDH).

Total proteins were extracted from cells using lysis buffer supplemented with the complete protease inhibitor cocktail (Abcam). The following primary antibodies were used for Western blot analysis: anti‐PTBP1 (ab133734, Abcam), anti‐SLC31A1 (ab129067, Abcam), anti‐Cleaved Parp antibody (ab32064, Abcam), anti‐Cleaved Caspase‐3 antibody (ab2302, Abcam), anti‐Bax antibody (ab32503, Abcam), anti‐Bcl‐2 antibody (ab32124, Abcam) and anti‐β‐actin antibody (ab8226, Abcam). The detailed steps of the assay have been described in our previous paper.³³ Briefly, the protein concentration was determined by BCA method, and then, total protein lysates were fractionated using SDS‐PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non‐fat milk and incubated with the above primary antibodies. Horseradish peroxidase‐conjugated secondary antibody was used to develop blots. The grey value was measured by ImageJ software.