Anti hnrnp a1

Antibodies were purchased from the following companies: anti-GLA from Santa Cruz; anti-PP1, Akt, phospho-PP1 at Thr³²⁰, phospho-Akt at Ser⁴⁷³, phospho-Akt at Thr³⁰⁸ from Cell Signaling Technology; anti-hnRNP A1 from Sigma; anti-hnRNP A2B1 from Acris; anti-SRp20 and SF2/ASF from Thermo Scientific; anti-H3K4me3, anti-H3K9me3, anti-H3K27me3, anti-H3K36me3, anti-H3K9me3, anti-H3K9A, anti-H3K9A, anti-H3K23A, anti-H3K27A, anti-H3S10P, anti-histone H3, anti-actin, and anti-HSP70 from Abcam; anti-p54nrb/NONO from Affinity Bioreagents; anti-PSIP1 from Bethyl Laboratories.

Antibodies used in this study include anti-TcFAZ antibody against a T. cruzi protein localized at the FAZ (prepared in house, manuscript in preparation), as well commercial antibodies as follow: Anti-SC35 (#S4045 - mouse), anti-GAPDH (#PLA0125 - rabbit), anti-U2AF35 (#SAB1300700 - rabbit), anti-hnRNPA1 (#R4528 - mouse), hnRNPA2B1 (#R4653 - mouse), and were from Sigma, USA. Other commercial antibodies included anti-RNA polymerase II - 8WG16, H5 and H14 (#MPY-127R - mouse, Covance, USA), anti-mouse IgM-HRP (#sc-2064 - goat, Santa Cruz, USA), biotin IgM conjugated secondary antibody (#553406 – rat, BD, USA), Streptavidin-HRP (#554066, BD).

Protein was extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Scientific). Proteins were separated on a 4–12 % Bis-Tris gel using the NuPAGE SDS Gel System (Life Technologies) and membranes were probed with anti-T7-tag antibody (AK42, Cold Spring Harbor Laboratory), anti-hnRNP A1 (R9778, Sigma-Aldrich), anti-hnRNP H (sc-10042, Santa Cruz Biotechnology), anti-TDP-43 (10782-2-AP, Proteintech Group), or anti-HPRT (HPA006360, Sigma-Aldrich).

The proteins were extracted from tissues or cells using RIPA lysis buffer (50 mM Tris, pH8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 1% sodium deoxycholate, 0.1% SDS) and analyzed in SDS-PAGE as the following description: after electrophoresis, the proteins were transferred onto nitrocellulose membrane (GE Healthcare). The membranes were blocked with 5% skim milk solution in TBST for one hour. The membranes were exposed to the primary antibody in 1% skim milk solution for one hour before they were washed three times for ten minutes with TBST, and then incubated for one hour with HRP-conjugated secondary antibody. After washing three times with TBST, the membranes were immersed in the ECL plus substrate (Amersham) for five minutes. The chemiluminescence signals were detected by X-ray films and analyzed by LabWorks Image Analysis Software. The primary antibodies used were anti-SMN (BD; 1:5000 dilution), anti-TRA2B (Abcam; 1:1000 dilution), anti-SF2/ASF (Zymed Laboratories Inc.; 1:1000 dilution), anti-hnRNP A1 (9H10, SIGMA; 1:1000 dilution), anti-hnRNP Q (Abcam; 1:1000 dilution), anti-SFRS9 (Abcam; 1:1000 dilution), and anti-Actin (Santa Cruz Biotechnology, Inc.; 1:3000 dilution). The secondary antibodies used were horse anti-mouse IgG-HRP (Cell Signaling; 1:5000 dilution), and donkey anti-goat IgG-HRP (Santa Cruz Biotechnology, Inc.; 1:5000 dilution).