Rabbit anti nf κb p65

Cells at 25% confluency were cultured in 6-well plates with coverslips for 24 h. The cells were fixed with 4% paraformaldehyde (Solarbio Technology Co., Ltd. Beijing, China) at 25 °C for 30 min, quenched with 30mM glycine (BGI, Shenzhen, China) in phosphate buffer saline (PBS, Boster Biological Technology Co., Ltd. Wuhan, China) at 25°C for 5min, and then permeabilized with 0.5% Triton-X (Solarbio) in PBS at 25°C for 5min. Pre-blocking was done with 5% non-fat milk and 2% bovine serum albumin (BSA, Gen-View Scientific Inc. USA) at 25°C for 1 h, and then the following primary antibodies were used to blot the specific proteins: rabbit anti-NF-κB p65 (1: 5000, Abcam), incubated with Fluor^® 488 goat anti-rabbit antibody(1: 500, ZSGB-BIO, Beijing, China) at 25°C for 1 h. Staining was done with 4′, 6-diamidin-2-phenylindole (DAPI), and observed with a confocal laser scanning microscope (Olympus Corporation, Beijing, China).

Total proteins were extracted from cell lysate with RIPA buffer (Thermo) and then quantified with a BCA protein assay kit (Beyotime), according to the specifications. Nuclear and cytoplasmic proteins were loaded into 10% sodium dodecyl sulfate (SDS) polyacrylamide gel, separately, and then transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore Corporation, Shanghai, China). After blocking with 5% non-fat milk, following primary antibodies were used to blot the special proteins at 4°C overnight: rabbit anti-IKKα/β pS180/S181 (1: 10000, Abcam), rabbit anti-NF-κB p65 (1: 5000, Abcam), rabbit NF-κB p65 pS536 (1: 10000, Abcam), rabbit anti-IκBα pS32 (1: 10000, Abcam), rabbit anti-NF-κB p100/p52 pS866 (1: 2000, Abcam), mouse anti-BRAF (F-7) (1: 200, Santa Cruz Biotechnology, Inc. Shanghai, China), rabbit anti-RET (1: 1000, Abcam), mouse anti-β-actin (1: 1,000, Proteintech Group, Inc. Wuhan, China), mouse anti-α-tublin (1: 10000, Proteintech). Goat anti-mouse and goat anti-rabbit secondary antibodies (1: 3000, Jackson Immuno Research Laboratories, Inc. West Grove, PA, USA) were used to incubate the proteins at 25°C for 1 h. An electrochemiluminescence (ECL, Millipore) system was used to expose the proteins.

Proteins were extracted from A549 cells using radioimmunoprecipitation assay buffer with protease inhibitor cocktail (both Cell Signaling Technology, Inc.). Protein concentration was determined using a BCA kit (CoWin Biosciences). The proteins (15 µg/lane) were separated using 10% SDS-PAGE and transferred to nitrocellulose membranes (Amersham; Cytiva). The membranes were subsequently blocked with 5% skim milk at room temperature for 2 h. Proteins were detected by western blotting using rabbit anti-HMGB1 (1:400; cat. no. ab18256; Abcam), rabbit anti-Toll-like receptor 4 (TLR4; 1:400; cat. no. ab13556; Abcam), rabbit anti-NF-κB p65(1:400; cat. no. ab207297; Abcam), and rabbit anti-GAPDH antibodies (1:1,000; cat. no. ab8245; Abcam) and were incubated at 4˚C overnight. The membranes were then incubated with the relevant goat anti-rabbit HRP-conjugated secondary antibodies (1:5,000; cat. no. ab7090; Abcam) at room temperature for 1 h, and the protein bands were visualized using ECL reagents (Amersham; Cytiva) and quantified by densitometry (ImageJ software; National Institutes of Health; http://rsbweb.nih.gov).

Cells
were washed twice with ice-cold
1× PBS and lysed with RIPA lysis buffer containing 1 mM phenylmethylsulfonyl
fluoride, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1
μg/mL pepstatin for 30 min on ice. The whole cell lysates were
centrifuged at 12,000g for 15 min at 4 °C, and
then, total protein concentration was determined using a BCA Protein
Assay Kit (Solarbio, China). The protein samples were electrophoresed
in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and
transferred to poly(vinylidene difluoride) membranes. After blocked
with 5% non-fat dry milk in PBS-T for 1 h at room temperature, the
membrane was incubated with the primary antibody overnight. After
three 10 min washes in PBS-T, the membranes were incubated with HRP-conjugated
secondary antibodies for 1 h at room temperature. After another three
10 min washes in PBS-T, immunoreactive proteins were detected with
ECL Western blotting analysis system (Solarbio, China) and captured
using ImageQuant LAS 500 imager (GE, USA).
The primary antibodies
used were as follows: rabbit anti-LC3 A/B (Cell Signaling Technology,
USA), rabbit anti-p62 (Abcam, USA), rabbit anti-IKK β (Abcam,
USA), rabbit anti-IκB α (Abcam, USA), rabbit anti-p-IκB
α (Cell Signaling Technology, USA), mouse anti-β-actin
(Santa Cruz, USA), rabbit anti-NF-κB p65 (Abcam, USA), mouse
anti-caspase 3 (Santa Cruz, USA), and rabbit anti-cleaved caspase
3 (Abcam, USA).