Mouse anti nfkb

Proteins from untreated and MPs-treated hGFs (for 48 h) were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis (Bio-Rad V3 Western Workflow™, Milan, Italy). Membranes were saturated for 120 min at room temperature in a blocking buffer (1 × TBS, 5% milk, 0.1% Tween-20) followed by overnight incubation at 4 °C with the following primary antibodies: mouse anti-NFkB (1:500; Santa Cruz Biotechnology), mouse anti-MyD88 (1:500; Santa Cruz Biotechnology) and mouse anti-NLRP3 (3 µg/mL; Novus). Subsequently, membranes were incubated for 60 min at room temperature with peroxidase-conjugated anti-mouse secondary antibody (1:5000; Bethyl Laboratories, Montgomery, AL, USA) [35 (link)]. Enhanced chemiluminescence with the Alliance 2.7 system (Uvitec Ltd., Cambridge, UK) was used to identify and quantify the bands obtained.

Firstly, the cells were washed twice with PBS prior to sample preparation. RIPA lysis solution (P0013C, Beyotime, Shanghai, China) was used to lyse the cells at 4 °C for 30 min. A BCA kit (P0010S, Beyotime, Shanghai, China) was used to detect the total protein concentration. Then, SDS–PAGE loading buffer (P0015F, Beyotime, Shanghai, China) was added to the lysate, and the mixture was boiled at 100 °C for 5 min. Then, 10–20 µg of protein per sample was separated with SDS-PAGE gels. Next, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA, ISEQ00010). The blots were incubated overnight at 4 °C with the corresponding primary antibodies and incubated with secondary antibodies at room temperature for 1 h, and developed with ECL substrate solutions A and B (Pierce™, Thermo Fischer). The primary antibodies used were: mouse anti-CDK4(sc-23896, Santa Cruz), mouse anti-Cyclin D1(sc-8396, Santa Cruz), mouse anti-p53(sc-126, Santa Cruz), mouse anti-BCL-2(sc-7382, Santa Cruz), mouse anti-BAX (sc-7480, Santa Cruz), mouse anti-NF-kB (sc-8008, Santa Cruz), mouse anti-Caspase3(sc-7272, Santa Cruz), and mouse anti-β-actin (sc-47778, Santa Cruz).

The following are other antibodies used in this study: anti-actin-HRP (horseradish peroxidase) (Santa Cruz, sc-47778), mouse anti-PCNA (Abcam, ab29), rabbit anti-XPO1-C-terminal (Santa Cruz, sc-5595), mouse anti-XPO1-N-terminal (Santa Cruz, sc-136220), mouse anti-Coilin (Sigma C-1862), mouse anti-XPO5 (Abcam ab57491), mouse anti-XPOT (Abcam ab49933), rabbit anti-H3 (Abcam ab1791), mouse anti-GEMIN5 (Santa Cruz, sc-136200), mouse anti-SMN1 (Abcam, ab5831), goat anti-LaminB (Santa Cruz, sc-6216), mouse anti-CSE1L (Santa Cruz sc-135855), rabbit anti-Dyskerin (Santa Cruz sc-48794), rabbit anti-PHAX (Bethyl, A303–916A), goat anti-PHAX (M-19) (Santa Cruz, sc-11704), mouse anti-UBF1 (Santa Cruz sc-13125), mouse anti-nucleolin (Enzo, ADI-KAM-CP100) mouse anti-TRF2 (Imgenex, IG124A), mouse anti-XPO5 (Abcam ab57491), mouse anti-NFKB (Santa Cruz sc-372), mouse anti-RANBP2 (Santa Cruz, sc-74518), mouse anti-Fibrillarin (Abcam, ab4566), mouse anti-FLAG M2 (Sigma), IgG mouse (Santa Cruz sc-2025), mouse anti-Coilin (Sigma, C1862), rabbit anti-TCAB1 (Novus, NB100–68252); IgG rabbit (Abcam ab37415). All Alexa-conjugated secondary antibodies were purchased from Life Technologies.