Ab154791

The A549 cells were seeded in six-well plate (5 × 10⁵ per well). After 24 h, either miCtrl or miR (final concentration at 50 nM) were transfected with lipofectamine 2000, respectively. Two days later, the cell lysates were harvested and the total protein was extracted with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The concentration of total proteins was measured with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts (30 µg total proteins), either from miCtrl or miR treated cells, were loaded and separated on SDS-PAGE gel electrophoresis. The proteins were transferred to membrane and incubated with antibodies (Anti-ILF2 antibody, 1:3000, ab154791; anti-Securin antibody, 1:5000, ab79546; anti-PTGR1 antibody, 1:2000, ab181131; anti-AKR1C1/AKR1C2 antibody, 1:3000, ab179448; anti-PCNA antibody, 1:3000, ab92552; anti-Peroxiredoxin 3 antibody, 1:10000, ab128953; anti-GAPDH antibody, 1:10000, ab128915; Abcam, Cambridge, MA, USA). After incubation with secondary antibody (Goat Anti-Rabbit IgG H&L (HRP), 1:4000, ab97051, Abcam), the images were acquired by using chemiluminesence detection.

The proteins collected from the radioimmunoprecipitation (RIPA) lysis buffer (Millipore) were subjected to separation with the use of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and moved to polyvinylidene fluoride (PVDF) membranes. The membranes were probed overnight with primary antibodies including SHMT2 (Abcam, ab155230, 1:5000), ILF2 (Abcam, ab154791, 1:1000), Bax (Abcam, ab32503, 1:1000), cleaved caspase-3 (Abcam, ab32042, 1:500), Bcl-2 (Abcam, ab32124, 1:1000), matrix metallopeptidase (MMP)2 (Abcam, ab92536, 1:1000), MMP9 (Abcam, ab76003, 1:1000), Vimentin (Abcam, ab92547, 1:1000), E-cadherin (Abcam, ab40772, 1:10,000), GAPDH (Abcam, ab9485, 1:2500) at 4°C following impeded with 5% nonfat milk for 1.5 h. Then, secondary antibody (Abcam, ab6721, 1:2000) was employed to incubate the membrane for 1 h at room temperature. The visualization of protein blots was presented with the employment of electrochemiluminescence (ECL) reagents (W1A003a, Wanleibio, Shenyang, China). Using Image J software (National Institutes of Health), protein levels were quantified.

The transfected cells were lysed on ice for 30 min in RIPA lysis buffer containing protease inhibitors. The lysed protein samples were added to 10 µl of pre-treated protein A agarose-coupled antibodies, including anti-IgG, anti-SHMT2 (Abcam, ab155230, 1:5000), or anti-ILF2 (Abcam, ab154791, 1:1000).