anti-β-actin antibody (#a3853)  (Millipore)

Journal: The Journal of Neuroscience

Article Title: Loss of Christianson Syndrome Na⁺/H⁺ Exchanger 6 (NHE6) Causes Abnormal Endosome Maturation and Trafficking Underlying Lysosome Dysfunction in Neurons

doi: 10.1523/jneurosci.1244-20.2021

Figure Lengend Snippet: Figure 12. Loss of NHE6 increases MVB fusion with the PM and exosome secretion. A, Representative TIRF image depicting MVB-PM fusion and exosome release as developed from Verweij et al. (2018). Widefield image of neuron cotransfected with mCherry and CD63-pHluorin expression constructs. White inset, Location of MVB-PM fusion and the zoomed in panels on the right. Each panel represents the progression of a CD63-pHluorin fusion event with the PM with the number of seconds indicated below the panel. Scale bars: large, 10mm; small, 1 mm. B, Quantification of full MVB-PM fusion/exosome release events per cell over 5 min in WT and Nhe6-/Y male littermate mouse primary hippocampal neurons at 14 DIV (WT n = 28 cells from 7 mice, Nhe6-/Y n = 18 cells from 7 mice, 5 litters, p = 0.009, Glass’s D = 2.27). C, Quantification of full MVB-PM fusion/exosome release events per cell over 5 min in WT and Nhe6-/Y male mouse primary hippocampal neurons at 14 DIV under the following conditions: untreated (same as in B), U18666A (positive control) (WT n = 14 cells from 5 mice, Nhe6-/Y n = 14 cells from 5 mice, 3 litters), bafilomycin A1 (positive control) (WT n = 14 cells from 7 mice, Nhe6-/Y n = 16 cells from 6 mice, 4 litters, Kruskal–Wallis test with Dunn’s test: WT untreated compared with WT bafilomycin A1 p = 0.002, Glass’s D = 3.04). D, E, CD63 western blot (D) and quantification (E) in WT and Nhe6-/Y male littermate mouse primary hippocampal neurons at 14 DIV (WT n = 5 cultures, Nhe6-/Y n = 5 cultures, 5 litters, p = 0.02, Glass’s D = 1.68). F, Released b -Hex enzyme activity following short-term incubation in Tyrode’s solution followed by treatment with either ionomycin or DMSO (WT n = 9, Nhe6-/Y n = 9, 8 litters). G, Released CatD enzyme activity following short-term incubation in Tyrode’s solution followed by treatment with either ionomycin or DMSO (WT n = 6, Nhe6-/Y n = 6, 5 litters). H, Released LDH activity across all b -Hex (WT n = 5, Nhe6-/Y n = 5, 5 litters) and CatD (WT n = 4, Nhe6-/Y n = 4, 3 litters) experiments. Data are mean 6 SEM. Unpaired two-tailed Student’s t test (C, WT-Nhe6-/Y: bafilomycin A1) with Welch’s correction (E), Mann–Whitney test (B,C, WT-Nhe6-/Y: U18666A), Kruskal–Wallis test with Dunn’s test (C, differences between treatments by genotype), two-way ANOVA with Tukey’s multiple comparisons test (F,G,H).

Article Snippet: The following antibodies were used for western blot: actin (Sigma, A3853, Ms, 1:1000), CatD (R&D Systems, AF1029, Gt, 1:1000), CD63 (Abcam, EPR21151-ab217345, Rb, 1:1000), ci-mannose 6-phosphate receptor (M6PR) (Cell Signaling Technology, 14364S, Rb, 1:500), GAPDH (Sigma, G8795, Ms, 1:40 000), LAMP1 (DSHB, 1D4B, Rt, 1:1000), RAB5 (Cell Signaling Technology, 3547, Rb, 1:1000), and RAB7 (Sigma, R8779, Ms, 1:1000).

Techniques: Expressing, Construct, Positive Control, Western Blot, Activity Assay, Incubation, Two Tailed Test, MANN-WHITNEY

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