Title: JNK activation is essential for activation of MEK/ERK signaling in IL-1β-induced COX-2 expression in synovial fibroblasts

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Christiana Gilbert
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1 Title: JNK activation is essential for activation of MEK/ERK signaling in -induced COX-2 expression in synovial fibroblasts Author list: Taku Kitanaka 1,a, Rei Nakano 1,a, Nanako Kitanaka 1, Taro Kimura 2, Ken Okabayashi 1, Takanori Narita 1 & Hiroshi Sugiya 1, *

2 Relative density of p-erk/t-erk Relative density of p-jnk/t-jnk Relative density of p-p38/t-p38 a b 60 * * 38 p-p38 38 t-p SB SB c d N.S. 30 * * SB p-jnk t-jnk 0 SB e p-erk1/2 t-erk1/2 f * N.S. * + + SB SB Fig. S1 The effect of the p38 inhibitor on the -induced phosphorylation of p38, JNK and ERK1/2. When the cells were pretreated with the p38 inhibitor SB (20 µm), the p38 inhibitor clearly attenuated the feline recombinant -induced p38 phosphorylation (a). The p38 inhibitor had no effect on the -induced phosphorylation of JNK (c) and ERK1/2 (e). Relative density of p-p38 (b), p-jnk (d) and p-erk (f) compared with that of the absent of is described. Results are presented as mean ± SE from 3 independent experiments. *P < 0.05.

SB239063 second row original figure 0 p-p38 + +

3 Figure S1a. first row original figure 0 p-p SB SB Figure S1a. second row original figure 0 p-p SB SB Fig. S1 (continue) Uncropped images for the blots shown in Figure S1a.

4 Figure S1c. first row original figure 0 p-jnk + + SB Figure S1c. second row original figure 0 t-jnk SB SB Fig. S1 (continue) Uncropped images for the blots shown in Figure S1c.

row original figure 0 t-erk1/2 + + SB239063

5 Figure S1e. first row original figure 0 p-erk1/2 Figure S1e. second row original figure 0 t-erk1/2 + + SB Fig. S1 (continue) Uncropped images for the blots shown in Figure S1e.

6 Relative density of p-p38/t-p38 Relative density of p-p38/t-p38 Relative density of p-p38/t-p38 a b N.S. 30 * * p-p-38 t-p PD PD c d N.S. 30 * * p-p38 t-p FR FR e f 30 * N.S. * p-p38 t-p SP6001 SP Fig. S2 The effect of the MEK, ERK or JNK inhibitor on the -induced phosphorylation of p38. A. The MEK inhibitor PD98059 (a; µm), the ERK inhibitor FR (c; µm) or the JNK inhibitor SP6001 (e; µm) had no effect on the IL- 1β-induced phosphorylation of p38. Relative density of p-p38 (b, d, f) compared with that of the absent of is described. Results are presented as mean ± SE from 3 independent experiments. *P < 0.05.

PD98059 second row original figure 0 t-p38 + +

7 Figure S2a. first row original figure 0 p-p PD PD98059 Figure S2a. second row original figure 0 t-p PD PD98059 Fig. S2 (continue) Uncropped images for the blots shown in Figure S2a.

FR180204 second row original figure 0 t-p38 + + + +

8 Figure S2c. first row original figure 0 p-p FR FR Figure S2c. second row original figure 0 t-p FR FR Fig. S2 (continue) Uncropped images for the blots shown in Figure S2c.

SP6001 second row original figure 0 t-p38 + + + +

9 Figure S2e. first row original figure 0 p-p SP6001 SP6001 Figure S2e. second row original figure 0 t-p SP6001 SP6001 Fig. S2 (continue) Uncropped images for the blots shown in Figure S2e.

(h) 0 12 24 48 Time (h) second row original

original figure 0 β-actin 0 12 24 48 Time (h) Fig.

10 Figure 1f. first row original figure 0 COX Time (h) Time (h) Figure 1f. second row original figure 0 COX Time (h) Figure 1f. third row original figure 0 β-actin Time (h) Fig. S3 Uncropped images for the blots shown in Figure 1f Time (h)

60120180 Time (min) Time (min) second row original figure 0

11 Figure 3a. first row original figure 0 p-mek Time (min) Time (min) Figure 3a. second row original figure 0 t-mek Time (min) Time (min) Fig. S4 Uncropped images for the blots shown in Figure 3a

Figure 3a. third row original figure 0 p-erk1/2 0 5 30 60120180 Time (min) Figure 3a.

12 Figure 3a. third row original figure 0 p-erk1/ Time (min) Figure 3a. fourth row original figure 0 t-erk1/ Time (min) Fig. S4 (continue) Uncropped images for the blots shown in Figure 3a.

(min) p-jnk sixth row original figure 0 0 5 30

13 Figure 3a. fifth row original figure Time (min) p-jnk Figure 3a. sixth row original figure Time (min) t-jnk Fig. S4 (continue) Uncropped images for the blots shown in Figure 3a.

30 60120180 Time (min) eighth row original figure 0 t-p38 0 5 30

14 Figure 3a. seventh row original figure 0 p-p Time (min) Time (min) Figure 3a. eighth row original figure 0 t-p Time (min) Time (min) Fig. S4 (continue) Uncropped images for the blots shown in Figure 3a.

15 Figure 4a. original figure 0 p-erk1/2 0 t-erk1/ PD98059 PD98059 Fig. S5 Uncropped images for the blots shown in Figure 4a.

16 Figure 4c. original figure 0 p-erk1/2 0 t-erk1/ FR FR Fig. S6 Uncropped images for the blots shown in Figure 4c.

17 Figure 4e. original figure 0 p-jnk PD98059 t-jnk PD98059 PD98059 Fig. S7 Uncropped images for the blots shown in Figure 4e.

18 Figure 4g. original figure 0 p-jnk 0 t-jnk + + FR Fig. S8 Uncropped images for the blots shown in Figure 4g.

19 Figure 5a. original figure 0 p-jnk 0 t-jnk + + SP6001 Fig. S9 Uncropped images for the blots shown in Figure 5a.

20 Figure 5c. original figure 0 p-mek 0 t-mek SP6001 Fig. S Uncropped images for the blots shown in Figure 5c. SP6001

21 Figure 5e. original figure 0 p-erk1/2 0 t-erk1/ SP6001 SP6001 Fig. S11 Uncropped images for the blots shown in Figure 5e.

22 Figure 6a. original figure IP: p-jnk IP: p-jnk IP: p-jnk 0 p-erk1/2 p-mek p-jnk 0 t-erk1/2 t-mek t-jnk IgG-LC + Fig. S12 Uncropped images for the blots shown in Figure 6a.

23 Figure 6b. original figure IP: p-mek IP: p-mek IP: p-mek 0 p-erk1/2 p-mek p-jnk 0 t-erk1/2 t-mek t-jnk IgG-LC + Fig. S13 Uncropped images for the blots shown in Figure 6b.

24 Figure 6c. original figure IP: p-erk IP: p-erk IP: p-erk 0 p-erk1/2 p-mek p-jnk 0 t-erk1/2 t-mek t-jnk IgG-LC + Fig. S14 Uncropped images for the blots shown in Figure 6c.

25 Figure 7a. original figure (bp) 200 β-actin JNK1 JNK2 JNK3 Fig. S Uncropped images for the blots shown in Figure 7a.

26 0 Figure 7c. first row original figure t-jnk1 Scramble JNK1 sirna JNK2 sirna Figure 7c. second row original figure 0 t-jnk2 Scramble JNK1 sirna JNK2 sirna Fig. S16 Uncropped images for the blots shown in Figure 7c.

27 Figure 7c. third row original figure 0 β-actin Scramble JNK1 sirna JNK2 sirna Fig. S16 (continue) Uncropped images for the blots shown in Figure 7c.

sirna + + + second row original figure 0 t-mek +

28 Figure 7f. first row original figure 0 p-mek + Scramble + + JNK1 sirna Figure 7f. second row original figure 0 t-mek + Scramble + + JNK1 sirna Fig. S17 Uncropped images for the blots shown in Figure 7f.

29 Figure 7f. third row original figure 0 p-erk1/2 + Scramble + + JNK1 sirna + Figure 7f. fourth row original figure Scramble + + JNK1 sirna t-erk1/2 + Scramble + + JNK1 sirna Fig. S17 (continue) Uncropped images for the blots shown in Figure 7f.

30 Figure 7f. fifth row original figure 0 t-jnk1 + Scramble + + JNK1 sirna Fig. S17 (continue) Uncropped images for the blots shown in Figure 7f.

31 Table S1 Primers used for RT-PCR or real-time RT-PCR Gene Name Gene bank ID Primer sequences COX-1 XM_ F: 5'-GGACAACCTGGAACGTCAGT-3' R: 5'-AAACACCTCCTGACCCACAG-3' COX-2 EF F: 5'-AACAGGAGCATCCAGAATGG-3' R: 5'-GCAGCTCTGGGTCAAACTTC-3' JNK1 XM_ F: 5'-AACTCTTTGACGCTGCTTGC-3' R: 5'-TGAAGCACTGTGCCTTTACC-3' JNK2 XM_ F: 5'-AGCCCAAGGGATTGTTTGTG-3' R: 5'-AGGACGAGTTCACGATAAGCTC-3' JNK3 XM_ F: 5'-TTTGTGCTGCGTATGATGCC-3' R: 5'-TTGGCATGCGTTTGGTTCTG-3' β-actin AB F: 5'-CTCTTCCAGCCTTCCTTCCT-3' R: 5'- GACAGCACCGTGTTAGCGTA-3' Table S2 Sequences for sirna transfection Gene Name Gene bank ID sirna sequences JNK1 XM_ F: 5'-GAAUCAGACUCAUGCUAAA-3' R: 5'-TTTAGCATGAGTCTGATTC-3' JNK2 XM_ F: 5'-GGUAUUAUCGGGCACCCGA-3' R: 5'-UCGGGUGCCCGAUAAUACC-3'

Osmotic stress induces the phosphorylation of WNK4 Ser575 via the p38mapk-mk pathway. Tokyo, Hongo, Bunkyo-ku, Tokyo , Japan

Osmotic stress induces the phosphorylation of WNK4 Ser575 via the p38mapk-mk pathway Junichi Maruyama 1, 2, Yumie Kobayashi 1, Tsuyoshi Umeda 1, Alain Vandewalle 3, Kohsuke Takeda 1, 4, Hidenori Ichijo