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. Author manuscript; available in PMC: 2019 Mar 1.
Published in final edited form as: Brain Behav Immun. 2017 Nov 13;69:139–153. doi: 10.1016/j.bbi.2017.11.008

Fig. 3. CRF amplification loop sustains activated TLR4 signal; α2 contribution.

Fig. 3

(A) Protein extracts from mock- or TLR4-transfected SK-N-SH cells (n=5 each) were immunoblotted with pCREB antibody, and the blots were sequentially stripped and immunoblotted with antibodies to pPKA, pERK1/2, TLR4 and β-Actin used as gel loading control. The results were quantitated by densitometric scanning and expressed as densitometric units normalized to β-Actin ± SEM. The levels of pCREB, pPKA and TLR4, but not pERK1/2, are significantly higher in the TLR4- than mock-transfected SK-N-SH cells (**p <0.01 by ANOVA). (B) Protein extracts from mock- or TLR4-transfected SK-N-SH cells treated or not (n=5 each) with the PKA inhibitor H89 (10 µM) were immunoblotted with antibody to pCREB and the blots were sequentially stripped and immunoblotted with antibodies to CRF (Bioss Antibodies, Cat. # bs-0246R), CRFR1 and β-Actin. The results are expressed as densitometric units normalized to β-Actin ± SEM. The levels of pCREB, CRF and CRFR1 are significantly higher in the TLR4- than mock-transfected cells, and upregulation is inhibited by H89 (*p <0.05; **p<0.01; *** p<0.001 by ANOVA). (C) Protein extracts from mock- or α2-transfected Neuro2a cells in the presence or absence of amplicons that deliver TLR4 (siTLR4) or scrambled (siNC) siRNA (n=5 each) were immunoblotted with antibody to CRF (Bioss Antibodies, Cat. # bs-0246R), the blots were sequentially stripped and immunoblotted with antibodies to TLR4 and β-Actin. Results are expressed as densitometric units normalized to β-Actin ± SEM. The levels of CRF and TLR4 are significantly higher in the α2- than mock-transfected cells and upregulation is inhibited by siTLR4, but not siNC (*p≤0.05 by ANOVA). (D) Neuro2a cells mock- or α2-transfected in the presence or absence of amplicons for TLR4 siRNA (siTLR4), α2 siRNA [siLA2 (Liu et al., 2011)] or scrambled siRNA (siNC) were stained with pCREB antibody (red) and examined for nuclear localization (activation). DAPI (blue) was used as nuclear counterstain. pCREB nuclear staining was minimal in the mock-transfected cells (CTL) and it was almost entirely cytoplasmic. α2 transfection significantly increased the % cells with nuclear pCREB staining and nuclear localization was inhibited by siTLR4 and siLA2, but not siNC (*** p≤0.001 by ANOVA).