anti pp65  (Cell Signaling Technology Inc)

Journal: Access Microbiology

Article Title: p62 limits Salmonella Typhimurium in macrophages through its role in cell signalling

doi: 10.1099/acmi.0.001102.v3

Figure Lengend Snippet: Depletion of p62 reduces the pro-inflammatory response of the macrophages. Relative fold change in IL-1⍺/β, IL1R1 (left), TNF⍺, IL-6 and IL-10 (right) gene expression, calculated by the 𝝙𝝙Ct method in control (Scrm) and p62 KD macrophages at 5 hpi (a) and 24 hpi (b) with S. Tm WT at an MOI of 5. A** = P <0.01, Student’s t-test. Data from three independent experiments, mean± sem . (c) ELISAs were performed to measure the protein concentrations of IL-1β (left) and TNF⍺ (right) in the supernatant of WT and p62 KD macrophages infected with WT S. Tm at an MOI of 5. * P <0.05, ** P <0.01, Student’s t-test. Data show the average of three independent experiments, mean± sem . (d) Supernatant at 5 hpi was replaced with media from previously infected WT iBMDMs (WTI), p62 KD iBMDMs (KDI) or conditioned media. Lysates were plated onto LB agar at the marked time points and colonies counted. MOI = 5, ns = not significant, ** P < 0.01, *** P <0.001, **** P <0.0001, Student’s t-test. Data show the average of three independent experiments, mean± sem . (e) Infected (MOI 5) and uninfected cell samples were lysed at 2 hpi and run through a 12% polyacrylamide gel to separate the proteins. (Left) The PVDF membrane was stained with an A20 antibody, representative of six independent experiments. (Right) Quantification of protein expression. * P < 0.05, *** P <0.001, Student’s t-test. Data show the average of six independent experiments, mean± sem . (f) Infected (MOI 5) cell samples were lysed at 15- and 30-min post-infection and run through a 12% polyacrylamide gel to separate the proteins. (Left) The PVDF membrane was stained with an anti-p65 and an anti-pp65, with actin as a loading control. Representative of three independent experiments. (Right) Quantification of protein expression, the percentage of pp65 in the total amount of p65. ** P < 0.01, Student’s t-test. hpi, hours post-infection.

Article Snippet: ⍺-p65 and pp65 , Cell Signalling , Rabbit/mouse , 1:1,000.

Techniques: Gene Expression, Control, Infection, Membrane, Staining, Expressing