rabbit polyclonal anti arf1  (Proteintech)

Journal: Cell Death & Disease

Article Title: BIG1 controls macrophage pro-inflammatory responses through ARF3-mediated PI(4,5)P2 synthesis

doi: 10.1038/s41419-020-2590-1

Figure Lengend Snippet: a WT and BIG1 −/− BMDMs were stimulated with LPS (100 ng/ml) for 30 min, the total, membrane-bounded and cytosolic proteins were measured by Western blot with the indicated antibodies. b Cell lysates from WT BMDMs transfected with Myc-ARF1 or Myc-ARF3 were subjected to pull-down assay, followed by western blot with indicated antibodies. c Cell lysates from WT BMDMs with or without LPS (100 ng/ml) treatment for 30 min were subjected to immunoprecipitation with BIG1 antibody, followed by Western blot with the indicated antibodies. d WT and BIG1 −/− BMDMs were treated with LPS (100 ng/ml) for 30 min, the activated ARF3 (ARF3-GTP) was pulled down by GST-GGA and visualized by Western blot. The densitometry of ARF3-GTP was quantified by Quantity One software and the activity of ARF3 in WT and BIG1 −/− BMDMs was measured. e WT BMDMs transfected with Si-ARF3 or negative control siRNA were treated with or without LPS (100 ng/ml) for 30 min. Total lysates were subjected to Western blot with indicated antibodies. f WT and BIG1 −/− BMDMs were transfected with Si-ARF3 or negative control siRNA for 48 h before treated with LPS for 6 h. The mRNA levels of TNF-α, IL-6, and IL-1β were measured by RT-qPCR. g , h BIG1 −/− BMDMs transfected with active mutant ARF3 (Q71L) or vector were treated with or without LPS (100 ng/ml). Total lysates were subjected to Western blot after treatment for 30 min ( g ), and the mRNA levels of TNF-α, IL-6, and IL-1β were measured by RT-qPCR after treatment for 6 h ( h ). i , THP-1-derived macrophages transfected with negative control siRNA (NC) or si-ARF3 were treated with or without LPS (100 ng/ml) for 30 min. The cell lysates were subjected to immunoblotting with the indicated antibodies. j THP-1-derived macrophages transfected with negative control siRNA or si-ARF3 were treated without or with LPS (100 ng/ml) for 6 h. The expression of TNF-α, IL-6, and IL-1β mRNA were measured by RT-qPCR. Data show pooled technical replicates from three independent experiments ( d , f , h , j ). All immunoblot data are representative of three independent experiments with similar results. Data are shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t -test in f and j ; one way ANOVA in d , h ).

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-β-Actin (12262, 1:1000, Cell Signaling), mouse monoclonal anti-GAPDH (51332, 1:1000, Cell Signaling), rabbit polyclonal anti-BIG1 (A300-998A, 1:1000, Bethyl), rabbit monoclonal anti-IκBα (4814, 1:1000, Cell Signaling), rabbit monoclonal anti-pS536-p65 (3033, 1:1000, Cell Signaling), rabbit monoclonal anti-pS176/180-IKKα/β (2697, 1:1000, Cell Signaling), rabbit monoclonal anti-TLR4 (14358, 1:1000, Cell Signaling), rabbit monoclonal anti-MyD88 (4283, 1:1000, Cell Signaling), rabbit polyclonal anti-TIRAP (ab17218, 1:1000, Abcam), mouse monoclonal anti-PIP2 (sc53412, 1:200, Santa Cruz), rabbit polyclonal anti-ARF1 (PA1-127, 1:2000, Thermo Fisher), mouse monoclonal anti-ARF3 (610784, 1:500, BD Biosciences), mouse monoclonal anti-ARF5 (H00000381-M01, 1:500, Abnova), rabbit polyclonal anti-ARF6 (ab77581, 1:1000, Abcam), and mouse monoclonal anti-Myc-Tag (2276, 1:1000, Cell Signaling).

Techniques: Western Blot, Transfection, Pull Down Assay, Immunoprecipitation, Software, Activity Assay, Negative Control, Quantitative RT-PCR, Mutagenesis, Plasmid Preparation, Derivative Assay, Expressing