Mouse anti cdk7

Proteins were detected using the following primary antibodies: anti-enterovirus VP1 clone 5-D8/1 antibody purchased from Dako (Denmark); Mouse anti-Ub, Mouse anti-MAT1, Mouse anti-CDK7, and Mouse anti-Cyclin H antibodies purchased from Santa Cruz (USA); Mouse monoclonal to RNA polymerase II CTD and Rabbit monoclonal to RNA polymerase II CTD (phosphor S5) antibodies purchased from Abcam (USA); Mouse anti β-actin purchased from Proteintech; Rabbit monoclonal CDK2 and CDK4, Rabbit monoclonal phosphor-CDK2 and CDK4, Mouse monoclonal pRb, Rabbit monoclonal pRb-phospho Ser780, pRb-phospho Ser795 and pRb-phospho Ser807/811 antibodies purchased from Cell Signaling (USA); Rabbit polyclonal Lamin B1, Mouse monoclonal c-Myc, Mouse monoclonal HA-Tag and Mouse Monoclonal Flag antibodies purchased from Sigma (USA); Goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody and goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody purchased from Thermo Fisher Scientific (USA).

For all IF stainings, primary neurons on coverslips were fixed for 15 min in 4% paraformaldehyde (PFA) at room temperature. Cells were permeabilised and blocked in PBS containing 10% donkey serum (SouthernBiotech, 0030-01) and 0,1% Triton X-100 for (108603 Merck Millipore) for 1 h at room temperature during gentle shaking. Incubation with primary antibodies in PBS-Triton 0.1% with 5% donkey serum was performed overnight at 4 °C in the following dilutions: mouse monoclonal anti-β-III tubulin (Tuj1) (1:1000; BioLegend # 801202), rabbit anti-cleaved caspase-3 (1:1000; Cell Signaling Technology # 9661), and mouse anti-Cdk7 (1:100, Santa Cruz # sc-7344). After three washing steps with PBS, secondary antibodies (Alexa Fluor^TM, Invitrogen) were used 1:1000 in PBS with 5% donkey serum and 0.1% Triton X-100 for 1 h at room temperature. Cells were washed two more times with PBS, counterstained for 10 min with DAPI 1:5000 (D9542 Sigma), and mounted with fluorescence mounting medium (Dako, S3023) before confocal microscopy (Olympus FV1000).