The CK2 inhibitor did not affect the eIF2α-CHOP arm of the ER stress-induced UPR.

<p>(A) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 4 h. Western blotting was then performed. TBB did not affect ER stress-induced eIF2α phosphorylation. Densitometric analysis of the normalization data of the phospho-eIF2α and eIF2α intensities were done. n = 3 per group. (B) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 6 h. A RT-PCR analysis was then performed. n = 3/group TBB did not affect ER stress-induced CHOP mRNA expression. (C) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 18 h. Western blotting was then performed. n = 4/group TBB did not affect ER stress-induced CHOP protein production. Results are expressed as the means ± S.E.</p

A CK2 inhibitor inhibited ER stress-induced GRP78 expression.

<p>4,5,6,7-tetrabromobenzotriazole (TBB) inhibited GRP78 expression at the mRNA and protein levels. (A) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and then treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 6 h. RT-PCR was performed using specific primers for each mRNA. n = 4/group *<i>p</i><0.05 compared with ER stress (Tm, Tg) alone. (B) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h, then treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 18 h, and subjected to Western blotting. n = 3/group *<i>p</i><0.05 compared with ER stress (Tm) alone. Results are expressed as the means ± S.E.</p

CK2 inhibitor did not affect ER stress-induced ATF6 processing.

<p>(A) SH-SY5Y cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h, and then treated with thapsigargin (Tg: 10 µM) for 18 h and Western blotting analysis was performed. TBB inhibited ER stress-induced GRP78 expression. *<i>p</i><0.05 (B) SH-SY5Y cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h, and then treated with thapsigargin (Tg: 10 µM) for 4 h and Western blotting analysis was performed using ATF6 antibody. (C) Densitometric analysis of p90ATF6. (D) Densitometric analysis of p50 ATF6. TBB did not affect ER stress-induced ATF6 cleavage. n = 2–3/group. Results are expressed as the means ± S.E.</p

Existence of CK2 at the endoplasmic reticulum.

<p>Primary cultured glial cells were immunostained for anti-CK2 (left, red) and anti-KDEL (A marker of ER; middle, green) antibodies, respectively. The right panel is a merged image of the left and middle panels. Scale bar, 10 µm.</p

ER stress did not affect expression of CK2.

<p>(A) Primary cultured glial cells were treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 18 h and subjected to Western blotting. ER stress increased GRP78 levels whereas it did not affect levels of CK2. (B) Densitometric analysis of expression of CK2. n = 4/group. Results are expressed as the means ± S.E.</p

Knocking down CK2 inhibited ER stress-induced GRP78 expression.

<p>(A) Western blot analysis of endogenous CK2α expression in lysates of nontransfected cells (NT) and cells transfected with (short interfering RNA) siRNAs (75 nM) directed at CK2 or the control sequence. CK2 siRNA reduced the expression of CK2 compared with NT or control siRNA. (B) CK2 siRNA decreased ER stress-induced GRP78 expression compared with control siRNA. Primary cultured glial cells were transfected with 75 nM siRNA and treated with tunicamycin (Tm: 0.01 µg/mL) for 4 h. RT-PCR was then performed. n = 3/group (C, D) CK2 siRNA decreased ER stress-induced GRP78 expression compared with control siRNA. Primary cultured glial cells were transfected with 75 nM siRNA, treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 18 h, and subjected to Western blotting. n = 3–4/group *<i>p</i><0.05, **<i>p</i><0.01 compared with control siRNA. Results are expressed as the means ± S.E.</p

Knocking down CK2 inhibited ER stress-induced XBP-1 splicing.

<p>CK2 siRNA decreased ER stress-induced XBP-1 splicing compared with control siRNA. Primary cultured glial cells were transfected with 75 nM siRNA and treated with tunicamycin (Tm: 0.01 µg/mL) for 4 h. A RT-PCR analysis was then performed. n = 3/group. Results are expressed as the means ± S.E.</p

The CK2 inhibitor inhibited ER stress-induced XBP-1 mRNA splicing.

<p>(A) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h, treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 6 h, and subjected to a RT-PCR analysis. (B) Densitometric analysis of spliced/unspliced XBP-1 mRNA. An ER stress inducer increased XBP-1 splicing and this effect was significantly inhibited by TBB. n = 4–5/group *<i>p</i><0.05 compared with ER stress (Tm, Tg) alone (C) Densitometric analysis of total XBP-1 mRNA (unspliced and spliced XBP-1 mRNA). n = 5/group. TBB did not affect the XBP-1 transcript. Results are expressed as the means ± S.E.</p