Prmt5 antibodies

Chromatin Immunoprecipitation (ChIP) assays were performed with K562 cells as described previously (28 (link)). Antibodies against histone H3K9ac and H4R3me2s were purchased from Abcam. PRMT5 antibodies were purchased from Sigma. Normal rabbit IgG served as the control. Precipitated DNA was analyzed by Q-PCR. ChIP samples were analyzed by quantitative real-time PCR using the FastStart Universal SYBR Green Master (Roche). A standard curve was prepared for each set of primers using serial titration of the input DNA. The percentage of ChIP DNA was calculated relative to the input DNA from primer-specific standard curves using the Rotor-Gene 6000 Series Software 1.7. ChIP primers are listed in Supplementary Figure S2. Student's t-test was used to derive the significance of the differences between mean values.

NCI-H929 cells and U266 cells were cultured on glass coverslips, fixed in 4% paraformaldehyde (Sigma) for 30 min and permeabilized using 0.3% Triton X-100 for 30 min at room temperature. After blocking with 5% goat serum in PBS, cells were incubated with PRMT5 antibodies (Sigma) for 60 min and secondary antibodies for 45 min, respectively, in a dark and humid chamber. Then, the cells were washed with PBS, and the nuclei were stained with 4,6,diarnidino-2-phenylindole (DAPI) for 5–10 min. Immunofluorescence images were captured under a fluorescence microscope (BX53, Olympus).

Bone marrow samples of newly diagnosed MM patients were collected at Nanjing Drum Tower Hospital, Jiangsu Province, China. MM patients with a distinctive pathologic diagnosis, no preoperative systemic or local treatment, and complete follow-up data were selected for inclusion in this study. The paraffin-embedded tissue were deparaffinized, rehydrated, and then subjected to antigen retrieval. The tissue slides were, respectively, incubated with PRMT5 antibodies (1:200; Sigma) and CASP1 antibodies (1:200; CST) overnight at 4 °C. Subsequently, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Each sample was independently scored by two pathologists who were blinded to all the patient clinical data. Based on the percentage of PRMT5- or CASP1-positive tumor cells, the extent of staining was scored 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). The final score for each slide was assessed by multiplying the scores for intensity and extent of staining. PRMT5 or CASP1 expression on slides was considered low if the score was <3 and high if the score was >3.