Amersham hybond ecl membrane

All the chemicals used for the various assays were of molecular reagent grade and were obtained from Sigma Aldrich (USA), Merck (India), SRL (India). The primary antibodies ERK ½, Phosho ERK ½, p38, and Phospho p38 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). JNK and phospho-JNK were obtained from Pierce (Thermo Scientific, USA). The peroxidase-conjugated secondary antibodies were from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The nitrocellulose membrane and CL–Xposure films were from Amersham (Amersham Hybond-ECL membrane, GE Healthcare, Little Chalfont, Buckinghamshire, UK). The enhanced chemiluminescence substrate (ECL) was from Pierce, West Pico Super Signal (Thermo Fisher Scientific, Marietta, USA).

Cells were lyzed in protein lysis buffer (2% SDS, 100 mmol/L Tris‐HCl, pH 6.8) and protein concentration was determined by a BCA assay (Pierce). Samples (60 μg) were denatured in sample buffer and were size separated using Bis‐Tris precast 4%–12% gels (Bio‐Rad, Hercules, CA, USA). The proteins were blotted onto an Amersham Hybond‐ECL membrane (GE Healthcare, Fairfield, CT, USA) for 45 min at 15 V. Membranes were blocked in 5% dry milk in PBS with 0.1% Tween (PBST) for 1 h at room temperature and incubated at 4°C with primary antibody in PBST with 0.5% dry milk overnight. The primary antibodies used in this study were rabbit monoclonal RABMAB αPTBP1 (Abcam, Cambridge, UK), rabbit αSRSF3 (Assay Biotech, Sunnyvale, CA, USA), rabbit αGAPDH (Cell Signaling, Danvers, MA, USA), and rabbit αActin (Sigma‐Aldrich). Membranes were washed 3 × 10 min in PBST and incubated for 1 h at room temperature with a 1:10,000 dilution of secondary HRP‐conjugated αrabbit antibody (Pierce) in 0.5% dry milk in PBST. Membranes were washed 3 × 10 min in PBST and the proteins bands were visualized using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA, USA) with Amersham Hyperfilm ECL (GE Healthcare). For SRSF3 band quantification, each band was firstly normalized to its respective Actin band and then related to its untreated control.

U2OS cells were harvested in lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl pH 8.0; Sigma-Aldrich) supplemented with 1x PIC-C (Calbiochem), incubated on ice for an hour, then centrifuged (13,000 rpm at 4 °C for 5 minutes). The supernatant lysates were mixed with the same amount of 2x SDS loading buffer containing 5% β-mercaptoethanol (Sigma-Aldrich) and boiled for 5 minutes. The lysates were separated in SDS-PAGE, transferred to Amersham Hybond ECL-membrane (GE Healthcare) and incubated with the following primary antibodies: anti-p53 (Dako, IS616), anti-S15 P p53 (Cell signalling, 9284), 1BP7G5 anti-RPB1 (from L. Tora, IGBMC), anti-S2P RPB1 (Abcam, ab5095) and anti-S5P RPB1 (Abcam, ab5131), anti-GAPDH (Millipore, MAB374); then the following secondary antibodies: RAM-HRP (Dako, P0260) and GAR-HRP (Dako, P0448). Chemiluminescent detection was conducted using Immobilon Western Chemiluminescent HRP substrate (Millipore).

Parasites at 5–10% parasitemia were harvested at 8-h intervals, erythrocytes were lysed in 0.075% saponin and parasite pellets then dissolved in 2 × reducing Laemmli buffer (125 mM Tris HCl pH 6.8, 20% glycerol, 4% SDS, 0.005% bromophenol blue, 5% beta-mercaptoethanol) at a concentration of 5 × 10⁶ infected erythrocytes/μl. Reduced parasite lysates were denatured at 95 °C for 5 min and approximately 1 × 10⁷ infected erythrocytes were separated by SDS-PAGE on NuPAGE® Novex® 4–12% Bis–Tris protein gel (Life technologies, NP0323BOX) in 1 × NuPAGE® MES SDS running buffer (Life technologies, NP0002) at 150 V for 70 min. Proteins were transferred to an Amersham HYBOND-ECL membrane (GE Healthcare, 45-000-929) in cold transfer buffer (192 mM glycine, 25 mM Tris, 3.5 mM SDS, 20% methanol) at 400 mA for 1 h and successful transfer was verified by Ponceau S (0.1% Ponceau S, 5% acetic acid) staining. The membranes were incubated with primary and secondary antibodies and bound antibodies were detected using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, WBKLS0500).

Total protein extracts from cell lines (15 μg each) were separated in a 10% SDS-PAGE and transferred into a nitrocellulose Amersham Hybond-ECL membrane (GE Healthcare). Rabbit polyclonal antibodies anti-NAPRT (HPA023739) from Sigma (St. Louis, MO, USA) and anti-PBEF (H-300) from Santa Cruz Biotechnology (Santa Cruz, CA, USA) were used at a 1:1000 dilution in 1% non-fat milk PBS Tween-20. Mouse monoclonal anti-α-tubulin antibody (Sigma) was used at a dilution of 1:40000 to assess protein loading. Goat anti-rabbit and goat anti-mouse horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) were used at 1:5000, and developed with the ECL-Plus detection kit (GE Healthcare).