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Fig. 3 | Journal of Neuroinflammation

Fig. 3

From: Reciprocal signals between microglia and neurons regulate α-synuclein secretion by exophagy through a neuronal cJUN-N-terminal kinase-signaling axis

Fig. 3

JNK2 or JNK3 shRNA reduce while JNK activation increases α-SNC secretion from neurons. a PC12 cells expressing α-SNC/p25α and JNK1, JNK2, or JNK1+2 shRNA were lysed after 48 h transgene induction and whole cell lysates and conditioned media (TCA) were subjected to western blotting against JNK1, JNK2, and α-SNC. Showing representative images from two to three independent experiments (we performed only two experiments with JNK1+2 shRNA expressing cells). b Differentiated SH-SY5Y cells expressing α-SNC/p25α and JNK1-3 shRNA were lysed after 48-h transgene induction, and whole cell lysates and conditioned media (TCA) were western blotted as indicated. The blots shown are representative of three independent experiments. c Differentiated SH-SY5Y cells expressing α-SNC/p25α were transfected with plasmids encoding constitutively active (FLAG-MKK7B2-JNK1-3) JNK isoforms or a kinase dead version of JNK1 (AFP) as control. The day after transfection, whole cell lysates and conditioned media (TCA) were western blotted as indicated. The blots shown are representative of three independent experiments. d Quantification of a α-SNC present in conditioned medium. Data are shown as mean + SEM (N = 3) percent relative to control transfected with scrambled (Scr) shRNA (*p < 0.05; ***p < 0.001 compared to “Scrambled”). e Quantification of b α-SNC present in conditioned medium. Data are shown as mean + SEM (N = 3) percent relative to control transfected cells (**: p < 0.01 compared to “Scramble”). f-h Quantification of (C) α-SNC present in conditioned medium (TCA), endogenous (Endo) p-JNK, and FLAG-MKK7B2-JNK1-3 (FLAG) p-JNK. Data are presented as mean + SEM (N = 3) normalized to control cells transfected with an empty vector (*p < 0.05; **p < 0.01; ***p < 0.001 compared to “Control”). i Calculated ratio of p-JNK of endogenous to FLAG-MKK7B2-JNK1-3 densitometric western blot signals

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