Anti rabbit starbright blue 700

Proteins from WCs, detergent-extracted CSK and flagella, or co-IPs were separated on SDS–PAGE gels. Separated proteins were transferred by semi-dry transfer to PVDF(polyvinylidene difluoride) and low-fluorescence PVDF membranes for further processing, as described (Albisetti et al., 2017 (link)). The antibodies used and their dilutions are listed in Supplementary file 7. For IP analysis, primary antibodies – anti-TY1 and rabbit anti-HA (GTX115044) – and fluorescent secondary antibodies – anti-mouse Starbright Blue 520 (Bio-Rad 12005867) and anti-rabbit Starbright Blue 700 (Bio-Rad 12004162) – were used.

VSMCs were stimulated with DOX (500 and 1,000 nmol/l) and Il-1β (100 ng/ml) in cell culture medium for 48 h. For protein extraction, cells were washed with ice-cold PBS and lysed in cold RIPA buffer (Thermo Fisher Scientific). Protein content was assessed with BCA protein assay kit (Pierce). 15–10 μg protein per lane (p21: 15 μg, all others 10 μg) was applied on the respective gel. Protein samples were mixed and dissolved in 4× Laemmli buffer (Biorad) and heated to 95°C for 15 min. The proteins were separated on 12% TGX Gels (Biorad) and transferred onto a polyvinylidene difluoride membrane. The membranes were immunoblotted overnight at 4°C with primary antibodies: rabbit anti-p21 (1:2,500, ab109199, Abcam), rabbit anti-Alp (1:500, 7H11L3, Invitrogen), rabbit anti-Cbfa1 (1:1,1000, sc-10758, Santa Cruz), and mouse anti-β-actin (1:5,000 8H10D10, Cell Signaling Technology). After washing five times for five min each with TBST, the membranes were incubated with conjugated fluorescent secondary antibodies [anti-rabbit-StarBright Blue700 (1:2,500, #12004162, Biorad) and anti-mouse-StarBright Blue520 (1:2,500, #12005867)]. The bands were visualized using a ChemiDoc MP Imaging System (Biorad).