Espada_et_al_2025_RIP Western blots
Description
The Western blots in the file are the original blots included in the Supplementary Figure S8. RNA Immunoprecipitation of proteins identified as potential lncRNAs interactors in S1m pull-down. Five proteins (Lb1, Lb2, Lb3, Lb4 and Lb5) were selected for RIP assays based on the S1m pull-down results. Immunoprecipitation was done using anti-HA magnetic beads and confirmed by western blotting using anti-HA antibody. Each membrane contains the total cell lysate, the input material (cell lysate after centrifugation for debris removal), the flow through (FT), the wash 1 (W1) and wash 7 (W7) and the eluates from the samples treated (Elution RNAse) or not (Elution) with RNAse.
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Steps to reproduce
Protein/RNA immunoprecipitation Immunoprecipitations were conducted as described previously by Walrad 64,65 with modifications. Briefly 1x109 log-phase promastigotes were harvested, washed twice with PBS and resuspended in 1mL of IP-Lysis Buffer 66. This material was immediately frozen in liquid nitrogen, defrosted and sonicated for 2 minutes twice (or until most of the parasites were lysed) and centrifuged 10,000 × g for 4 min at 4 °C. Supernatant was then incubated with 1mg of Pierce™ Anti-HA Magnetic Beads (Thermo Scientific™) for 2 hours at 4 °C under rotation. For the negative control (untagged parental cell line), magnetic beads were blocked with 100 μg of HA peptide for 30 minutes at 4 °C under rotation in IP-Lysis Buffer. Magnetic beads were washed at least 7 times to remove unbound proteins and divided in two tubes. Half of the material was destined to RNA extraction and DNAse treated as described in “RNA extraction section”. The other half was further divided in two tubes. One tube was incubated with 2x Laemmli buffer at 95°C for five minutes to extract the proteins from the beads. The other half was treated with 25 μg of DNAse and protease-free RNAse (Thermo Scientific™) in IP-Lysis Buffer without RNAse inhibitor by incubation at 22°C for 15 minutes. Magnetic beads were then washed three more times and incubated with 2x Laemmli buffer at 95°C for five minutes. The presence of the tagged protein in the eluate material was evaluated by immunoblotting using 1:5000 anti-HA antibody (Sigma-Aldrich) in PBST-1% milk solution for 1 hour at 4°C, followed by incubation with 1:10,000 anti-Rabbit IgG (Promega) in PBST-1% milk solution overnight at 4°C. Membranes were incubated with Amersham ECL Prime Western Blotting Detection Reagents (Cytiva) and exposure and acquisition were conducted in BioRad ChemiDoc Imaging System.
Institutions
- Universidade de Sao PauloSão Paulo, Sao Paulo