Anti nf κb p65 polyclonal antibody

An HFHSD (23.3% protein, 46% carbohydrate, and 20.4% fat) was purchased from Research Diets Company, D12327 (New Brunswick, NJ, USA).
Glucosamine (GLC; D-glucosamine hydrochloride, biotech grade, CAS number: 66-84-2) was purchased from Shanghai McLean Biochemical Technology Co., Ltd. (Shanghai, China).
Metformin was purchased from Sino-US Shanghai Squibb Pharmaceutical Co., Ltd. (CAS number: ABH5474) (Shanghai, China).
The primary antibodies used were an anti-TLR4 monoclonal antibody (66350-1-Ig, 1:1000), anti-CD14 monoclonal antibody (60253-1-Ig, 1:1000), anti-MYD88 monoclonal antibody (67969-1-Ig, 1:1000), anti-GAPDH polyclonal antibody (10494-1-AP, 1:5000), anti-ZO-1 polyclonal antibody (21773-1-AP, 1:100), anti-Occludin polyclonal antibody (27260-1-AP, 1:100), and anti-Claudin-1 polyclonal antibody (13050-1-AP, 1:500). The above antibodies were purchased from Proteintech (Wuhan, China). An anti-NF-κB p65 polyclonal antibody (ab16502, 1:1000) and anti-NF-κB p65 (phosphor-S536) polyclonal antibody (ab76302, 1:1000) were purchased from Abcam Company (Cambridge, UK).

The protein extracts from HaCaT cells treated by PM_2.5 were obtained by cell lysis buffer (Beyotime, China). Total proteins for each sample were loaded onto a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. After electrophoresis, proteins were transferred onto a PVDF membrane. After being blocked for 2 h at room temperature in 5% BSA (Beyotime, China), the PVDF membrane was incubated with primary antibodies over night at 4 °C.
The following primary antibodies were used in this research: anti-NLRP1 rabbit monoclonal antibody (1:1000, Cell Signaling Technology), anti-GAPDH mouse monoclonal antibody (1:1000, Proteintech), anti-IL-1-β rabbit monoclonal antibody (1:1000, Cell Signaling Technology), anti-NF-κB p65 polyclonal antibody (1:5000, Abcam). Then, the membrane was rinsed three times in TBST (10 min each at room temperature), and incubated for 1 h at room temperature with a HRP-linked secondary antibody of corresponding species (anti-rabbit IgG, 1:3000, Cell Signaling Technology; anti-mouse IgG, 1:3000, Cell Signaling Technology) and finally scanned with a LAS3000 imaging system (Fujifilm, Tokyo, Japan) after Immobilon Western (Millipore, USA) infiltration.

An HFHSD (23.3% protein, 46% carbohydrate, and 20.4% fat) was purchased from Research Diets Company, D12327 (New Brunswick, NJ, USA).
Glucosamine (GLC; D-glucosamine hydrochloride, biotech grade, CAS number: 66-84-2) was purchased from Shanghai McLean Biochemical Technology Co., Ltd. (Shanghai, China).
Metformin was purchased from Sino-US Shanghai Squibb Pharmaceutical Co., Ltd. (CAS number: ABH5474) (Shanghai, China).
The primary antibodies used were an anti-TLR4 monoclonal antibody (66350-1-Ig, 1:1000), anti-CD14 monoclonal antibody (60253-1-Ig, 1:1000), anti-MYD88 monoclonal antibody (67969-1-Ig, 1:1000), anti-GAPDH polyclonal antibody (10494-1-AP, 1:5000), anti-ZO-1 polyclonal antibody (21773-1-AP, 1:100), anti-Occludin polyclonal antibody (27260-1-AP, 1:100), and anti-Claudin-1 polyclonal antibody (13050-1-AP, 1:500). The above antibodies were purchased from Proteintech (Wuhan, China). An anti-NF-κB p65 polyclonal antibody (ab16502, 1:1000) and anti-NF-κB p65 (phosphor-S536) polyclonal antibody (ab76302, 1:1000) were purchased from Abcam Company (Cambridge, UK).