Anti dicer

Cells were harvested in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% deoxycholate, and a protease inhibitor cocktail (Roche, Basel, Switzerland)). The lysates were then subjected to 12% SDS-PAGE and Western blotting according to our standard procedures [18 (link),44 (link)]. The antibodies used in this study included anti-tubulin (1:3000), anti-His (1:10,000), anti-Flag (1:5000), and anti-Dicer (1:2000), which were purchased from Proteintech, Rosemont, IL, USA. The JAK1/JAK2 inhibitor Ruxolitinib (INCB018424) was purchased from Selleck, Houston, TX, USA (CAS No. 941678-49-5).

The total protein was isolated from tissues or cells using a total protein extraction kit (KeyGEN BioTECH, Nanjing, China) following the manufacturer's protocol. Equal amounts of protein were separated by 10% SDS-PAGE and then transferred onto PVDF membranes (Millipore, CA, USA). After blocking with 5% skim milk for 2 h at room temperature, the PVDF membranes were incubated first with specific primary antibodies at 4°C overnight and then with a secondary antibody at room temperature for 1 h. Finally, the signals on the membranes were visualized using an Odyssey CLx Infrared Imaging System (LI-COR Biosciences, NE, USA). The following antibodies were used in this study: anti-DHX9 (Proteintech, #17721-1-AP; 1:1000 dilution), anti-RUNX1 (Proteintech, #25315-1-AP; 1:1000 dilution), anti-KSRP (Bioworld Technology, #BS9961 M; 1:1000 dilution), anti-Flag (Affinity Biosciences, #T0053; 1:1000 dilution), anti-Drosha (ZEN-Bioscience, #381855; 1:1000 dilution), anti-Dicer (Proteintech, #20567-1-AP; 1:1000 dilution) and anti-GAPDH (Proteintech, #10494-1-AP; 1:5000 dilution).

The large TMA was constructed by contract service at the National Engineering Center for Biochip (Shanghai, China). Each array dot was punched to 1.5 mm diameter from the paraffin tumor block or the normal renal tissues. The standard protocol for IHC of TMA was used as described previously [30 (link)]. The polyclonal rabbit anti-Dicer (1:50, Proteintech, USA) and monoclonal rabbit anti-MMP-2 (1:50, Cell Signaling Technology, MA) were used for primary antibody incubation at 4°C overnight. The slides without primary antibody incubation were used as negative control.