Anti ptbp1 mouse monoclonal antibody

RIP was performed using the Abcam protocol (https://www.abcam.com/epigenetics/rna-immunoprecipitation-rip-protocol) with some modifications. HEK293T/17 cells were plated into 10-cm plates, followed by plasmid transfection described above. On the following day, the cells were irradiated with 300 mJ/cm² of 254 nm UV light, and nuclei and cytoplasmic fractions were isolated. Nuclear pellets were sheared by sonication with 5 cycles (ON for 30 s, OFF for 30 s) using a Bioruptor Pico device. To immunoprecipitate RNA with the antibodies for target proteins, each lysate was incubated at 4°C overnight. Protein A/G magnetic beads (Invitrogen) were added to bind the target antibodies, and sequential washing was conducted to remove unbound antibodies. The anti-hnRNP K mouse monoclonal antibody [3C2]-ChIP Grade (ab39975, Abcam) and anti-PTBP1 mouse monoclonal antibody (32-4800, Thermo Fisher Scientific) were used to immunoprecipitate HNRNPK and PTBP1, respectively, in each nuclear or cytoplasmic fraction. To purify the RNA, the lysates were incubated with protease K at 55°C overnight, followed by Trizol (Thermo Fisher Scientific) and chloroform (WAKO) extraction. RNA levels were quantified by RT-qPCR.

Cells were plated in twelve-well plates, transfected with plasmid(s), lysed in Cell Lysis buffer (Cell Signaling), and incubated at 4°C for 1 h. The cell lysates were applied to 10% precast polyacrylamide gels (Bio-Rad) for SDS-PAGE and transferred to nitrocellulose membranes (Amersham). The membranes were incubated for 1 h at room temperature with the primary antibody, anti-GFP rabbit polyclonal antibody (1:1000 dilution; A6455, Thermo Fisher Scientific), and then for 45 min at room temperature with the secondary antibody, anti-rabbit IgG conjugated with HRP (P0448, Dako), and EGFP was detected by ECL detection reagent (Amersham). As a control, anti-β actin mouse monoclonal antibody (1:1000 dilution; A5441, Sigma Aldrich) was used as the primary antibody, and anti-mouse IgG–conjugated HRP (1:1000 dilution; P0447, Dako) was used as the secondary antibody. To detect endogenous target proteins, anti-hnRNPK mouse monoclonal antibody [3C2]-ChIP Grade (ab39975, Abcam), anti-PTBP1 mouse monoclonal antibody (32-4800, Thermo Fisher Scientific), RPL7A rabbit polyclonal antibody (PA5-30155, Thermo Fisher Scientific), RPS3A rabbit polyclonal antibody (PA5-29398, Thermo Fisher Scientific), and anti-UCHL1 mouse monoclonal antibody (CL3210, Sigma Aldrich) were used at 1:1000 dilution with overnight incubation at 4°C.