Ab109451

Cos-7 cells were seeded onto 10-cm² dishes the day before transfection. The following plasmids psg5-V5-PRMT5, pcdna3.1-HA-HP1γ, and pcdna3.1-GR were transfected into Cos-7 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. 48 h after transfection, cells were treated (or not) with Dex for 24 h, and cell extracts were prepared in RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 0.25% deoxycholate) supplemented with protease inhibitor tablets and phosphatase inhibitors (1 mM NaF, 1 mM Na₃VO₄, and 1 mM β-glycerophosphate). Protein extracts were incubated with HP1γ primary antibody (ab10480; Abcam), PRMT5 (ab109451; Abcam) or GR/DGH2L (#12041; Cell signaling) over night at 4°C under agitation. Protein A Agarose (Millipore) beads were then added, and the mixture was incubated 2 h at 4°C. The immunoprecipitates were separated on SDS–PAGE. Immunoblotting was conducted with primary antibodies against GR G-5 (sc-393232; Santa Cruz), GR/D6H2L (#12041; Cell signaling), HP1γ (ab10480; Abcam), and PRMT5 (07-405; Millipore). Secondary antibodies were used for chemiluminescence detection using the ECL detection reagent (Roche Molecular Biochemicals) according to the manufacturer’s instructions. For immunoprecipitation experiments, 3% of the input of each sample was analyzed by immunoblot.

Total RNA was extracted using RNAiso Plus (Takara, China) according to the manufacturer’s protocol. Quantitative PCR analysis of PRMT5-ISO5 was carried out with 10 μL TB Green™ Premix Ex Taq™ II (Takara, China) by the QuantStudio 3 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The primers for RT-qPCR are shown in Supplementary Table S1.
Proteins extracted from cells and tissues were measured and heat denatured. Equal amounts of proteins were separated using 10% or 12% SDS-PAGE and transferred to PVDF membranes (Millipore, Boston, MA, USA). After saturating, the membranes were incubated with anti-PRMT5 (ab109451, Abcam, Boston, MA, USA), anti-HNRNPH1 (ab10374, Abcam, Boston, MA, USA), anti-SRSF3 (ab198291, Abcam, Boston, MA, USA), anti-AKT1 (75692S, CST, Boston, MA, USA), anti-phospho-AKT (Ser473) (4060S, CST, Boston, MA, USA), anti-SDMA (13222S, CST, Boston, MA, USA) or anti-GAPDH (60004-1-Ig, Proteintech, Shanghai, China), and subsequently incubated with HRP-conjugated secondary antibodies (Biosharp, Guangzhou, China). Finally, the membranes were detected using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and visualized with ChemiScope 6100 (CliNX Science Instruments, China).

Cells were fixed in 4 % paraformaldehyde for 15 min, washed twice in phosphate-buffered saline, and then incubated with 10 % goat serum at 37 °C for 15 min. Primary anti-Fragilis antibody (ab15592, Abcam, 1:200), anti-Mvh antibody (ab13840, Abcam, 1:100) or anti-Prmt5 (ab109451, Abcam, 1:50), and secondary antibody IgG (SA00003-2, Proteintech, 1:200) were used for immunostaining. Images were acquired using a Nikon A1Si confocal microscope or Leika DMI 3000 B inverted microscope.

The expression of PRMT5 and BTG2 proteins was assessed with immunohistochemistry in a tissue microarray using rabbit polyclonal antibodies specifically against PRMT5 (Abcam, Cambridge, USA ab109451), BTG2 (Abcam, Cambridge, USA ab85051), and Ki67 (Abcam, Cambridge, USA ab15580). Immunohistochemistry staining was performed using the Dako Envision Plus System (Dako, Carpinteria, CA) according to the manufacturer's protocol.
Each sample was independently scored by two pathologists who were blinded to all the patient clinical data. The intensity of staining was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). Based on the percentage of 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. The slide was considered negative if the score was <5 and positive if the score was above 5.