Page 420 - Read Online

Page 4 of 11 Davis et al. Neuroimmunol Neuroinflammation 2018;5:50 I http://dx.doi.org/10.20517/2347-8659.2018.60

TMEM119 expression was also assessed by fluorescent immunocytochemistry. Briefly, C20 cells were seeded
on glass coverslips in 6-well dishes and cultured as described above until 70%-80% confluent. Following
the treatment period (24 h), cells were washed three times with PBS then fixed in 4% paraformaldehyde for
15 min. Cells were washed with PBS and then incubated in blocking buffer (1% BSA in PBS) overnight at
room temperature. Cells were then incubated in anti-TMEM119 (1:500 in PBS containing 0.5% BSA, Sigma
#HPA051870) overnight at 4 °C with rocking. After washing with PBS, cells were incubated for 2 h in Alexa
Fluor® 488 donkey anti-rabbit antibody (1:3000 in PBS containing 0.5% BSA; Life Technologies #A-21206).
Cells were washed again with PBS, treated with 300 nmol/L 4′ ,6-diamidino-2-phenylindole (DAPI; Sigma)
without rocking for 15 min at room temperature, then washed in PBS prior to mounting in Prolong Gold
anti-fade reagent (Invitrogen; Eugene, OR).

Cytokine/chemokine expression
Levels of secreted CXCL10, CCL2, and IL-6 were measured in the culture medium using standard dual-
antibody solid phase immunoassay (ELISA) kits purchased from Peprotech. Cytokine/chemokine
concentrations were normalized to total cellular protein levels, which were determined using the
[50]
bicinchoninic acid protein assay as previously described .
Expression of inflammatory signaling molecules
Induction of inflammatory signaling was determined by measuring phosphorylation of inhibitory kappa
B alpha (IκBα), and p38 mitogen-activated protein kinase (p38 MAPK) in cytoplasmic fractions; and
phosphorylation of nuclear factor (NF)-κB p65 in nuclear fractions. More specifically, after experimental
treatments, cells in 100 mm dishes were washed twice with PBS and nuclear and cytoplasmic protein
[51]
extracts prepared as previously described , with the exception of including 1 mmol/L sodium
orthovanadate (Sigma, #450243) in the lysis buffers. Thirty micrograms of total protein were loaded on 7.5%
polyacrylamide gels (BioRad TGX FastCast Acrylamide kit, #161-0171), electrophoresed and then transferred
to PVDF membrane. Membranes were then incubated at 4 °C for 15-18 h with rocking. Primary antibodies
included anti-phospho-IκBα (1:100, Cell Signaling #2859S), anti-IκBα (1:1000, Cell Signaling #4812S), anti-
phospho-p38 (1:500, Cell Signaling #9215S), anti-p38 (1:1000, Cell Signaling #9212S), anti-phospho-NF-
κB p65 (1:1000, Cell Signaling #3033S), anti-NF-κB p65 (1:1000, Cell Signaling #4764S), and anti-β-tubulin
(1:1000, Cell Signaling #2146S). Anti-rabbit IgG, AP-linked (1:1000, Cell Signaling #7054S) was used as the
secondary antibody and restore western blot stripping buffer was used to remove antibodies and allow for re-
labelling of membranes. The blots were scanned and analyzed as described above in the section on microglia
marker expression.

Statistical analyses
Prism version 7.0 software (GraphPad Inc., San Diego, CA) was used for figure presentation and statistical
analysis. Analyses included one-way analysis of variance (ANOVA), followed by Tukey’s multiple
comparisons test. In those instances where data did not exhibit homogeneity of variance (P < 0.05 for
Bartlett’s test), data were log-transformed prior to analysis. Data are presented as mean ± SEM (n = 4-7) and
P < 0.05 indicated statistically significant differences between groups.

RESULTS
Expression of microglial marker
TMEM119 was robustly expressed in IL-1β-treated C20 cells as indicated by a strong band at ≈ 29 kDa [Figure
1 Top]. Whereas, TMEM119 was not detected in either unstimulated C20 cells or in NHA by western blot
analysis. As expected, the astrocyte marker, GFAP was expressed in NHA, but was not detected in C20 cells.

Using fluorescent immunocytochemistry we confirmed our western blot findings that IL-1β-treated C20
cells express TMEM119 [Figure 1 Bottom]. This immunocytochemistry approach revealed that TMEM119 is
also expressed in unstimulated C20 cells. In both unstimulated and IL-1β-stimulated cells, TMEM119 was