Fig 1: CaCl2treatment increases cell surface residence of ZIP8 and ZIP14 in hBMVECs.A, cells were pretreated with 2 mM CaCl2 or 10 μM BAPTA-AM in RPMI 1640 minus Ca2+, minus serum supplemented media for 3 h prior to cell-surface biotinylation of hBMVEC for ZIP8 and ZIP14. Arrows represent the monomeric form of the protein, and asterisks designate an oligomeric form of the protein. Fold change in band intensity was normalized to β-actin probed in the blot of input (total) protein. The total and biotinylated cell-surface protein (bound) was quantified for ZIP8 (B) and ZIP14 (C); n = 3 to 4 biological replicates per condition were performed each with n = 4 experimental replicates. The blots in panel A are representative of experimental replicates from one of the labeling experiments. Note that in this analysis, following probing for ZIP8, the blots were stripped and then probed for ZIP14. Statistical significance was determined using one-way ANOVA and Tukey’s multiple comparison test for input and surface protein groups separately, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. hBMVEC, human brain microvascular endothelial cell; ZIP8, ZRT IRT-like protein 8; ZIP14, ZRT IRT-like protein 14.
Fig 2: Cytoplasmic Ca2+induces the trafficking of ZIP8.A, hBMVECs grown on coverslips were treated with either 2 mM CaCl2 or RPMI 1640 minus Ca2+, minus serum media as an untreated control. Cells were fixed, stained with wheat germ agglutinin (WGA), blocked, and then incubated with rabbit primary antibodies against ZIP8 and ZIP14. Coverslips were incubated in anti-rabbit Alexa Fluor 488-conjugated secondary for 1 h followed by a 10-min nuclear stain with Hoechst 33342. Coverslips were mounted using Prolong Gold antifade mounting media and sealed. Images were acquired at 63 × magnification with oil immersion on a Leica TCS SP8 confocal microscope. Images were adjusted for brightness and quantified using ImageJ. B, perinuclear fluorescence represents pixel values around the nucleus, and nonperinuclear fluorescence represents values obtained by subtracting perinuclear fluorescence from whole-cell fluorescence. Both perinuclear and nonperinuclear measurements were divided by whole-cell measurements to obtain the percent of whole-cell values. For ROI quantification, n = 10 cells in seven separate fields per condition were analyzed. Statistical significance was determined using compared to untreated control, ns = not statistically significant, **p < 0.01. ZIP8, ZRT IRT-like protein 8; ZIP14, ZRT IRT-like protein 14.
Supplier Page from MilliporeSigma for Anti-ZIP8 antibody produced in rabbit