JNK1 Antibody (F-3)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lyse cells in lysis buffer (100 mm NaCl, 50 mm Tris-HCl, 1 mm EGTA, 10 mmMgCl2, pH 7.2) containing 1 % Triton X-100, phosphatase, and protease inhibitor mixture	Dilute primary Ab for western blotting (starting dilution 1:200, dilution range 1:100-1:1000)

Upstream tips
Lyse cells in lysis buffer (100 mm NaCl, 50 mm Tris-HCl, 1 mm EGTA, 10 mmMgCl2, pH 7.2) containing 1 % Triton X-100, phosphatase, and protease inhibitor mixture
Protocol tips
Dilute primary Ab for western blotting (starting dilution 1:200, dilution range 1:100-1:1000)

Publication protocol

Cells were lysed in lysis buffer (100 mm NaCl, 50 mm Tris-HCl, 1 mm EGTA, 10 mmMgCl2, pH 7.2) containing 1 % Triton X-100, phosphatase, and protease inhibitor mixture for 5 min at room temperature and thereafter kept on ice. Cell lysates were centrifuged at 16,100×g for 5 min at 4 °C, and protein concentrations of the supernatant were determined with Dc protein assay (Bio-Rad, Copenhagen, Denmark), before the addition of Laemmli buffer and loading of equivalent protein quantities on SDS-polyacrylamide gels. Following transfer to PVDF membranes, western blotting was performed using chemiluminescent HRP detection substrate (Millipore, Hellerup, Denmark). Specifically, for p-JNK in differentiated PC12 cells exposed to Ra2-conditioned medium (Fig. 6e, ​,f),f), Ra2 cells were changed to HBSS ± LPS (0.5 μg/ml) ± NGF for 6 h before conditioned HBSS was collected from Ra2 monoculture and centrifuged 6000 rpm at 4 °C for 3 min prior to transfer to differentiated PC12 cell monoculture for a 6-h incubation. After 6 h, PC12 conditioned medium was recovered and cells lysed and prepared for western blot as described. All western blot bands were quantified with ImageJ or Image Lab.

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