WesternBlots_PRO-IP-seq
Description
Original, scanned WB-films reported in the study: "PRO-IP-seq Tracks Molecular Modifications of Engaged Pol II Complexes at Nucleotide Resolution" by Vihervaara A, Versluis P and Lis JT. Pol II was detected with primary antibodies against the largest (RPB1) subunit of Pol II, and the films developed using HRP-conjugated secondary antibodies. Tubulin is shown as a loading control. Levels of Pol II CTD modifications were queried during a heat shock time course.
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Instant heat shock was conducted by pelleting K562 cells and re-diluting the cells into pre-warmed, preconditioned media. The cells were then placed as one batch in 42°C water bath. 1 million cells were collected at indicated heat shock time point, and the heat shock terminated by instantly pipetting the collected cells into ice-cold PBS, followed by two PBS washes. The cells were lysed in buffer C (25% glycerol, 20 mM HEPES pH 7.4, 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT), the soluble fraction collected, and protein concentration measured with Qubit. 20 μg of total soluble protein was loaded into SDS-PAGE and transferred into nitrocellulose membrane. The membranes were incubated with primary antibodies against Pol II RPB1 (polyclonal ab against the N-terminal domain, Santa Cruz, sc899), or specific modifications of RPB1 C-Terminal Domain (CTD): serine-2-phosphorylated CTD (Millipore, 04-1571), serine-5-phosphorylated Pol II CTD (Millipore, 04-1572), and serine-7-phosphorylated CTD (Millipore 04-1570). Since the target proteins (RBP1 in different modifications) reside in the same kDa range, each Pol II modification was blotted in a distinct membrane. β-tubulin (Abcam, ab6046) was visualized as loading control. The secondary antibodies were HRP conjugated (GE Healthcare), and the blots developed using an enhanced chemiluminescence method (ECL kit; GE Healthcare). Film for each membrane was scanned, and the area shown in the study is indicated with a dashed box. Westen Blot detection of HSF1 (ADI-SPA-901 antibody from Enzo Life Sciences, not shown in the manuscript) is included as a reference of the heat-shock; During heat shock, HSF1 gains phosphorylation which is detected as a slower migrating HSF1 protein in an SDS-PAGE gel. The longer the heat shock, the higher in the gel HSF1 resides. The HSF1 blot also serves to illustrate the under-loading of well 1 (non-heat shock) in membrane that was used to detect Pol II CTD serine-5-phosphorylation. Please note that levels of unphosphorylated Pol II CTD (8WG16, abcam) during heat shock were published earlier (Vihervaara et al., 2021, Mol Cell). The raw data for unphosphorylated Pol II CTD (8WG16) upon heat shock is reported here: https://doi.org/10.17632/gycj6tnw6v.1