Pvfd membrane

Cells were grown in 6-well plates. Before collection, the cells were washed with PBS and lysed in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40 (Igepal CA-630), 0.5% Na-deoxycholate, 0.1% SDS) supplemented with 2× protease inhibitor cocktail set (Merck Millipore, Germany). Proteins were separated in 5% (for Dicer detection) or 10% (for Tubulin detection) polyacrylamide gel and transferred to a PVFD membrane (Merck Millipore, Germany). Anti-Dicer (#349; [27 (link)]) and anti-Tubulin (#T6074, 1:5000; Sigma-Aldrich, USA) primary antibodies and HRP-conjugated secondary antibodies (1:50,000) were used for signal detection with SuperSignal West Femto chemiluminescent substrate (Pierce, Thermo Fisher Scientific, USA ).

Ice-cold PBS was employed to rinse the cells twice after harvesting. After that, cell lysis was performed using the RIPA lysis buffer as described by the manufacturer (Beyotime, Shanghai, China). Then, we utilized the BCA protein assay kit to quantify the proteins (Beyotime). After 5 × SDS-PAGE loading buffer (Beyotime) was added and mixed, protein samples were boiled for 5 min. Protein with the amount of 30 μg was successfully fractionated with a 10 % SDS-PAGE gel and blotted onto a PVFD membrane (Merck Millipore, Billerica, MA, USA). Subsequently, 3 % TBST solution containing 5 % dry milk was used to block the membranes at RT for 1 h. After that, we inoculated the membranes with anti-ApoM (F2051-1G9, Abnova, Taipei, Taiwan) and anti-RPS27A (#3936, Cell Signaling Technology, Boston, MA, USA) antibodies at 4 °C overnight. GAPDH (sc-32233, Santa Cruz, CA, USA) was employed as the standard for normalizing protein loading. After rinsing four times using TBST, the membranes were probed with HRP-conjugated AffiniPure goat anti-rabbit IgG (#7074, CST) or horse anti-mouse IgG (#7076, CST) at RT for 1.5 h. Afterwards, the Pierce ECL Western Blotting Substrate kit (Thermo Scientific, Rockford, IL, United States) was used to visualize protein bands after washing the membranes with TBST.

3T3 cells were grown in six-well plates. Before collection, the cells were washed with PBS and lysed in lysis buffer (20 mM Hepes [pH 7.8], 100 mM NaCl, 1 mM EDTA [pH 8.0], 0.5% IGEPAL-25%, 1 mM fresh DTT, 0.5 mM PMSF, 1 mM NaF, 0.2 mM Na₃VO₄, supplemented with 2× protease inhibitor cocktail set [Millipore], 2× phosphatase inhibitor cocktail set [Millipore], and RiboLock RNase inhibitor [Thermo Fisher Scientific]). Proteins were separated on 10% polyacrylamide gel and transferred to a PVFD membrane (Millipore). Anti-HA (11867431001, clone 3F10, rat, 1:2,500; Roche), anti-PKR (#ab32052, 1:5,000 dilution; Abcam), and anti-tubulin (#T6074, 1:5,000; Sigma-Aldrich) primary antibodies and anti-rat HRP (1:50,000) secondary antibody were used for signal detection with SuperSignal West Femto or Pico Chemiluminescent Substrate (Pierce).