Journal: Frontiers in immunology

Article Title: Mevalonate kinase-deficient THP-1 cells show a disease-characteristic pro-inflammatory phenotype.

doi: 10.3389/fimmu.2024.1379220

Figure Lengend Snippet: FIGURE 1 Characterization of WT, MKD-HIDS, MKD-MA and MKD-MA*MK THP-1 cells cultured for 24 hours under standard culture conditions. (A) Immunoblot analysis of MK, HMGCR, Rap1 and unprenylated Rap1a (uRap1a). (B) MK activity in pmol/(mg.min) indicated as mean ± SD of duplicate measurements.

Article Snippet: Membranes were blocked in 2% bovine serum albumin (BSA; Sigma) in TBS with 0.1% Tween-20 (TBS-T) for 1 hour at room temperature, followed by overnight incubation at 4°C with primary antibodies in 2% BSA in PBS-T. Primary antibodies were used against MK (1:5,000, own production (27), HMGCR (1:1,000, Atlas antibodies, AMAb90618), unprenylated Rap1a (1:500, Santa-Cruz, sc-373968), total Rap1 (1:500, Santa-Cruz, sc398755), SRC (1:1,000, Cell Signaling, 2110) and phosphorylated Src family kinases (Tyr416) (1:1,000, Cell Signaling, 2101).

Techniques: Cell Culture, Western Blot, Activity Assay

Journal: Frontiers in immunology

Article Title: Mevalonate kinase-deficient THP-1 cells show a disease-characteristic pro-inflammatory phenotype.

doi: 10.3389/fimmu.2024.1379220

Figure Lengend Snippet: FIGURE 2 Effect of culturing WT, MKD-HIDS and MKD-MA THP-1 cells for 24 hours in the presence of delipidated FBS or at elevated temperature. Immunoblot analysis of MK, HMGCR, Rap1 and unprenylated Rap1a (uRap1a) of THP-1 cell cultured at the indicated temperatures in standard medium with 10% FBS, or at 37°C in the presence of 10% delipidated FBS (D).

Article Snippet: Membranes were blocked in 2% bovine serum albumin (BSA; Sigma) in TBS with 0.1% Tween-20 (TBS-T) for 1 hour at room temperature, followed by overnight incubation at 4°C with primary antibodies in 2% BSA in PBS-T. Primary antibodies were used against MK (1:5,000, own production (27), HMGCR (1:1,000, Atlas antibodies, AMAb90618), unprenylated Rap1a (1:500, Santa-Cruz, sc-373968), total Rap1 (1:500, Santa-Cruz, sc398755), SRC (1:1,000, Cell Signaling, 2110) and phosphorylated Src family kinases (Tyr416) (1:1,000, Cell Signaling, 2101).

Techniques: Western Blot, Cell Culture

Journal: Frontiers in immunology

Article Title: Mevalonate kinase-deficient THP-1 cells show a disease-characteristic pro-inflammatory phenotype.

doi: 10.3389/fimmu.2024.1379220

Figure Lengend Snippet: FIGURE 5 Pharmacological rescue of unprenylated Rap1a (uRap1a) protein levels and IL-1b release. MKD-MA THP-1 cells were cultured for 20 hours in the presence of TAK-475 or GGOH and stimulated for an additional 4 hours with 10 ng/ml LPS. Immunoblot analysis of uRap1a, total Rap1 and tubulin in MKD-MA THP- 1 cells cultured in the presence of LPS and TAK-475 (A) or GGOH (C). uRap1a protein levels were quantified and expressed relative to the vehicle control treated MKD-MA THP- cells (B, D). IL-1b release was measured in the supernatants of WT and MKD-MA THP-1 cells cultured in the presence of TAK-475 (E) or GGOH (F). Data are presented as mean ± SD of at least two independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test, *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001.

Article Snippet: Membranes were blocked in 2% bovine serum albumin (BSA; Sigma) in TBS with 0.1% Tween-20 (TBS-T) for 1 hour at room temperature, followed by overnight incubation at 4°C with primary antibodies in 2% BSA in PBS-T. Primary antibodies were used against MK (1:5,000, own production (27), HMGCR (1:1,000, Atlas antibodies, AMAb90618), unprenylated Rap1a (1:500, Santa-Cruz, sc-373968), total Rap1 (1:500, Santa-Cruz, sc398755), SRC (1:1,000, Cell Signaling, 2110) and phosphorylated Src family kinases (Tyr416) (1:1,000, Cell Signaling, 2101).

Techniques: Cell Culture, Western Blot, Control, Comparison

Journal: Molecular brain

Article Title: Disruption of protein geranylgeranylation in the cerebellum causes cerebellar hypoplasia and ataxia via blocking granule cell progenitor proliferation.

doi: 10.1186/s13041-023-01010-4

Figure Lengend Snippet: Fig. 7 Decreased geranylgeranylation of RhoA and increased expression of p21 in Ggps1 cKO mice. A Western blotting for Ggpps and non-prenylated Rap1a. There was a significant reduction on Ggpps levels in Ggps1 cKO mice (**P = 0.010; n = 3 mice per group). There was a significant increase on protein levels of non-prenylated Rap1a in Ggps1 cKO mice compared with controls at P0 (**P = 5.3 × 10–4; n = 3 mice per group). β-actin was used as the internal control. Raw data were shown in Additional file 4: Fig. S4A. B Quantitative RT-PCR analysis on p21. There was a significant increase on p21 mRNA levels in Ggps1 cKO mice compared with controls at P0 (***P = 1.5 × 10–4; n = 5 mice per group per age). C Western blotting results for p21. There was a significant increase in Ggps1 cKO mice at P0 (**P = 0.001; n = 3 mice per group). Raw data were shown in Additional file 4: Fig. S4B. D Western blotting for Rho A. Protein lysates for total (T) cerebellum, membrane (M) and cytosol (C) fractionations were prepared from mice at P0. Raw data were shown in Additional file 4: Fig. S4C. E Relative levels of RhoA in different lysates at P0. There was no significant difference on levels of total RhoA between control and Ggps1 cKO mice (P > 0.05, n = 5 mice per group). There were significant differences on RhoA levels in the cytosol and membrane fractionations between control and Ggps1 cKO mice (*P = 0.010, *P = 0.039, respectively; n = 5 mice per group). β–actin and ATP1A1 were used as the internal control for the cytosol and membrane fractionations, respectively

Article Snippet: The membrane was blocked with 5% non-fat milk (w/v) for 1 h and incubated with one of the following primary antibodies overnight at 4 °C: Ggpps (Santa Cruz, sc-271680), Pax6 (Biolegend, 901301), Rap1A (Santa Cruz, sc-65), Rap1 (Santa Cruz, sc-398755), RhoA (Santa Cruz, sc-418), p21 (CST, 64016S), Atp1a1 (Proteintech, 55187-1-AP), β-actin (ABclonal, AC026).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Membrane