Ptbp1 antibody

RIP was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, 17-700) according to the manufacturer’s instructions. HUVECs in 15-cm dishes were collected and resuspended in cold RIP lysis buffer, and then stored at –80 °C overnight. Magnetic beads were preincubated with 1 µg of PTBP1 antibody (Invitrogen, 32-4800) or with 1 µg of control IgG (Millipore, 03-241). Next, the frozen homogenates were thawed quickly and centrifuged at 18,000 × g for 10 min, and 10 µl of the supernatant was stored as input. Then, for each RIP reaction, 100 µl of supernatant was incubated overnight with the magnetic bead-antibody complex at 4 °C. On the second day, the RNA/protein immunocomplex was extensively washed with RIP Wash Buffer (provided in the kit). The cross-linking was reversed by incubation with proteinase K. The immunoprecipitated RNA was purified through phenol:chloroform:isoamyl alcohol (125:24:1) isolation. The purified immunoprecipitated RNA was reverse-transcribed into cDNA using an RNA-to-cDNA Kit (Takara, RR036A). qRT-PCR was performed using this cDNA as a template to quantify the ARRB1 mRNA; the following primers were used: ARRB1 (forward: 5’-TACAGTCGTTCCCACCGG-3’ and reverse: 5’-GACGCACAGAATTCCGCT-3’) and GAPDH (forward: 5’-GTCTCCTCTGACTTCAACAGCG-3’ and reverse: 5’- ACCACCCTGTTGCTGTAGCCAA-3’).

Grow cells in 6-well plates containing confocal slides (1 × 10⁴ cells per well). After washing with PBS, cells were fixed with 4% paraformaldehyde for 15 min, and 0.5%Triton X-100 was permeable for 20 min. After the slides were soaked with PBS, 1%bovine serum albumin was blocked for 30 min, each slides were dripped with sufficient amount of diluted Paxillin antibody (Abcam, UK, Cat. no. ab32084) and incubated overnight at 4℃ in a wet box. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody (Abcam, UK, Cat. no. ab150077) was added in the dark and incubated in a wet box at 37℃. Then, cells were incubated overnight at 4℃ in a wet box with diluted PTBP1 antibody (Invitrogen, USA, Cat. no. 32-4800), followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) antibody (Abcam, UK, Cat. no. ab150115). Hoechst 33342 (Beyotime Biotechnology, China, Cat. no. C1025) was added to the drops and incubated for 5 min, and the specimens were nucleated. The tablets were sealed with a sealing solution containing an anti-fluorescence quench agent, and the acquired images were observed under a confocal laser scanning microscope (Carl Zeiss, Germany) at 200x magnification.