Expression of Brefeldin A (BFA)-treated cells, co-localization with cellular markers, and ER stress analysis of panx1a and the Y205A mutant. (A) Images of panx1a-EYFP transfected Neuro 2a cells treated with 5ug/mL BFA for 19h showed a reduction of cell membrane localization. Scale bar: 10µm. (B) Western blot analysis showed that treatment with 5ug/mL BFA for 19h caused a decrease in the Gly2 state and an increase in the Gly1 state of the wild type panx1a protein. No effect was detected for the Y205A mutant. Proteins were detected using an anti-GFP antibody. ß-actin served as a protein loading control. (C, D) Co-localization of EYFP-tagged panx1a and the Y205A mutant with mCherry-tagged Sec24D (COP II vesicle marker), His-tagged Cav-1 (Caveolin-1 marker), DsRed-tagged calreticulin (ER marker), and DsRed-tagged galactosyltransferases (Golgi apparatus marker). His-tagged Cav-1 was detected using an anti-His antibody and Alexa Fluor 568 secondary antibody. (E, F) Co-localization quantification of the wild type and mutant panx1a with the vesicle and organelle markers. Error bars show standard error of the mean. WT panx1a + Sec24D: n = 34, WT panx1a + Cav-1: n = 36, WT panx1a + ER: n = 29, WT panx1a + Golgi: n = 35, Y205A + Sec24D: n = 30, Y205A + Cav-1: n = 31, Y205A + ER: n = 20, Y205A + Golgi: n = 20. (G) Real-time qPCR analysis of ER stress genes. Neuro 2a cells were transfected with EYFP alone, panx1a-EYFP, and Y205A-EYFP. When indicated, cells were treated with 5ug/mL BFA for 19h. 18s rRNA was used as the reference gene. EYFP-transfected cells were used as the control group. Data were collected in three independent experiments in triplicate for each gene. All data were relative to the EYFP control group. ****p < 0.0001, ***p < 0.001, *p < 0.05.
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