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. 2022 Feb 25;50(5):2700–2718. doi: 10.1093/nar/gkac104

Figure 4.

Figure 4.

Microglial activation and non-canonical NF-κB signalling in ATM-deficient cells are mediated by NIK kinase. (A) Representative immunoblot analysis of the non-canonical NF-κB pathway proteins in whole-cell, cytoplasmic and nuclear extracts of NIK-deficient WT and ATM KO HMC3 microglia. Loading controls: GAPDH (whole-cell, cytoplasmic), PARP1 (nuclear). *Non-specific band. (B) RT-qPCR analysis of NIK knockdown efficiency in WT and ATM KO HMC3 microglia as in (A). Expression is relative to Ctrl siRNA-treated WT HMC3. Reference gene: RSP13. Mean ± S.D. shown (n = 3). Two-way ANOVA with Tukey's multiple comparison's test was used for 2-ΔCt values. C Quantification of protein levels as in (A). Data are relative to loading control. Mean ± S.D. shown (n = 3). Two-way ANOVA with Tukey's multiple comparison's test used (P-values are shown relative to WT Ctrl siRNA). (D) RT-qPCR analysis of the indicated cytokines as in (A). Expression is relative to Ctrl siRNA-treated ATM KO HMC3. Reference gene: RSP13. Mean ± S.D. shown (n = 4 except for IL6 where n = 3). Two-way ANOVA with Tukey's multiple comparison's test used. (E) Phagocytosis levels (5 μm beads) of cells treated as in (A). Phagocytosis is relative to Ctrl siRNA-treated WT HMC3, in which 3.5 ± 1.7% of cells are phagocytic. Mean ± S.D. shown (n = 4). Two-way ANOVA with Tukey's multiple comparison's test used.