mouse anti-β-actin a3853  (Millipore)

Journal: Cell Death & Disease

Article Title: Endotoxin stabilizes protein arginine methyltransferase 4 (PRMT4) protein triggering death of lung epithelia

doi: 10.1038/s41419-021-04115-7

Figure Lengend Snippet: A MLE12 cells were treated with LPS as indicated; cell lysates were immunoblotted with PRMT4 and β-actin antibodies. B BEAS-2B cells were treated with LPS as indicated; cell lysates were immunoblotted with PRMT4 and β-actin antibodies. C Primary human small airway epithelial cells (HSAECs) were treated with LPS as indicated; cell lysate was collected and analyzed with PRMT4 and β-actin antibodies. For A – C , relative expression of PRMT4 was plotted in the below panels. D Lung tissue lysates from deidentified human lung samples from normal and infected patients were analyzed for PRMT4 and β-actin by immunoblotting. Densitometry was presented in the right panel. E PRMT4 mRNA levels were determined by qRT-PCR in LPS-treated bronchial epithelial cells. Experiments n = 3. Statistical significance was indicated with asterisk “*” between groups where P < 0.05 vs. control (0).

Article Snippet: Cycloheximide (Cat#: ALX-380-269-G001, lot: 01061518) and Ubiquitin aldehyde (Cat#: BML-UW8450-0050, lot: 07021447) were from Enzo Life Sciences (Farmingdale, NY). β-Actin (Cat#: A3853) antibody and bacterial lipopolysaccharide (LPS) from E. coli O111:B4 (Cat#: L4391, lot: 115M4090V) were from Sigma (Carlsbad, CA).

Techniques: Expressing, Infection, Western Blot, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Endotoxin stabilizes protein arginine methyltransferase 4 (PRMT4) protein triggering death of lung epithelia

doi: 10.1038/s41419-021-04115-7

Figure Lengend Snippet: A BEAS-2B cells were treated with cycloheximide (CHX), leupeptin, or MG132 as indicated. Cell lysates were immunoblotted with PRMT4 and β-actin antibodies. B Densitometry results in A were analyzed in semi-log format with GraphPad prism 5 and the results were plotted. C Ectopic expression of ubiquitin reduces PRMT4 protein in a concentration-dependent manner. Densitometry was plotted in the right panel. D PRMT4 is polyubiquitinated as shown by co-immunoprecipitation (Co-IP) of precipitates analyzed with ubiquitin and PRMT4 antibodies.

Article Snippet: Cycloheximide (Cat#: ALX-380-269-G001, lot: 01061518) and Ubiquitin aldehyde (Cat#: BML-UW8450-0050, lot: 07021447) were from Enzo Life Sciences (Farmingdale, NY). β-Actin (Cat#: A3853) antibody and bacterial lipopolysaccharide (LPS) from E. coli O111:B4 (Cat#: L4391, lot: 115M4090V) were from Sigma (Carlsbad, CA).

Techniques: Expressing, Concentration Assay, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Cell Death & Disease

Article Title: Endotoxin stabilizes protein arginine methyltransferase 4 (PRMT4) protein triggering death of lung epithelia

doi: 10.1038/s41419-021-04115-7

Figure Lengend Snippet: A – C MLE12 cells, BEAS-2B cells, and human primary small airway epithelial cells (HSAECs) were treated with LPS as indicated. Cell lysates were analyzed by PRMT4, cleaved caspase 3, FBXO9, and β-actin immunoblotting. Shown below is densitometric analysis of immunoblots. D Overexpression of PRMT4 increases cleaved caspase 3, 8, and 9 baseline levels in BEAS-2B lung epithelial cells. E Knockdown of PRMT4 in BEAS-2B epithelial cells with shRNA reduces cleaved caspase 3, 8, and 9 expression. F , G Overexpression of PRMT4 enhances LPS-induced caspase 3 activation ( F ) and causes BEAS-2B lung epithelial cell death. Cell death is determined using LDH assay and the data are normalized with that from untreated control cells. H , I Silencing of PRMT4 inhibits LPS-induced caspase 3 activation ( H ) and cell death ( I ) in BEAS-2B cells. * P < 0.05. Experiments n = 3.

Article Snippet: Cycloheximide (Cat#: ALX-380-269-G001, lot: 01061518) and Ubiquitin aldehyde (Cat#: BML-UW8450-0050, lot: 07021447) were from Enzo Life Sciences (Farmingdale, NY). β-Actin (Cat#: A3853) antibody and bacterial lipopolysaccharide (LPS) from E. coli O111:B4 (Cat#: L4391, lot: 115M4090V) were from Sigma (Carlsbad, CA).

Techniques: Western Blot, Over Expression, shRNA, Expressing, Activation Assay, Lactate Dehydrogenase Assay