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. 2021 Aug 26;9(5):1357–1367. doi: 10.1016/j.gendis.2021.08.005

Figure 5.

Figure 5

MG132 inhibits the activation of NF-κB caused by RGS12. (A) BMMs were transfected with pCMV-RGS12 or pCMV control for 48 h and treated with MG132 for 12 h. The protein levels of pIκB, IκB, pNF-κB, NF-κB, and β-Actin were measured by immunoblotting assay. (B) The ratio of pIκB/IκB, pNF-κB/NF-κB, and IκB/β-Actin in (A) were calculated. β-Actin as an internal control. Note that the MG132 could inhibit the pIκB and pNF-κB activation induced by RGS12 in macrophages. Data are mean ± SEM. ∗∗∗P < 0.001 and ∗∗P < 0.01 versus WT group, (n = 3). (C, D) The RAW264.7 cells were transfected with pCMV (Control) (C) and pCMV-RGS12 (RGS12 OE) (D) for 24 h. Immunofluorescence showing that the nuclear translocation of NF-κB (p65) in control and RGS12 OE group (Red, NF-κB (p65); Green, α-Tubulin; Blue, DAPI; bar = 5 μm). Parts of the whole table showed the location of NF-κB (Blue, nucleus and cytoplasm, green, cytoplasm only and purple, nucleus only). (E) The RAW264.7 cells were transfected with pCMV-RGS12 (RGS12 OE) and added MG132 for 24 h. Immunofluorescence showing that the nuclear translocation of NF-κB (p65) in RGS12 OE with MG132 group (Red, NF-κB (p65); Green, α-Tubulin; Blue, DAPI; bar = 5 μm). The pie chart table showed the location of NF-κB (Blue: nucleus and cytoplasm, green: cytoplasm only and purple: nucleus only). (F, G) The RAW264.7 cells were transfected with pCMV (Control) or pCMV-RGS12 (RGS12 OE) with or without MG132 for 24 h. Immunoblots showing the expression of NF-κB in the nucleus and cytoplasm. Data are mean ± SEM. ∗∗∗P < 0.001, ∗∗P < 0.01, and ∗P < 0.05, (n = 3). (H) Real-time PCR analysis for the expression of TNFα, IL6, and IL1β in Control, RGS12 OE and RGS12 OE + MG132 groups as depicted in (F). Data are presented as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001 versus the control group (n = 5).