rabbit anti-impdh2 polyclonal antibody 12948-1-ap  (Proteintech)

Journal: The FEBS journal

Article Title: Y12C mutation disrupts IMPDH cytoophidia and alters cancer metabolism.

doi: 10.1111/febs.70086

Figure Lengend Snippet: Fig. 1. Introducing the Y12C point mutation into IMPDH1 and IMPDH2 in cultured HEK 293T cells using the ABEmax base editor. (A) Genomic sequences of human IMPDH1 and IMPDH2 at the Y12 regions and the corresponding sgRNA sequences. The target adenine (A) residues at positions A7 and A8, which are the intended base-editing sites, are indicated by arrows. (B) Diagram showing the strategy for establishing no-cytoophidium cell lines. (C) Immunofluorescence staining of IMPDH (green) and DAPI (magenta) in HEK 293T cells co- transfected with ABEmax-T2A-mCherry and sgRNA constructs targeting IMPDH1 or IMPDH2. Cells are treated with MPA (100 lM) for 1 h before fixation. Scale bars are 20 lm. Images are representative of n = 3 independent experiments performed in triplicate.

Article Snippet: The following antibodies were used: rabbit anti-IMPDH2 polyclonal antibody (1 : 10 000, 12948-1-AP, ProteinTech), rabbit anti-HPRT polyclonal antibody (1 : 5000, GTX113466, GeneTex, Irvine, CA, USA), HRP-conjugated mouse anti-b-ACTIN monoclonal antibody (1 : 3000, HRP60008, ProteinTech) and HRP-conjugated goat anti-rabbit IgG polyclonal antibodies (1 : 10 000, 31460, Invitrogen).

Techniques: Mutagenesis, Cell Culture, Genomic Sequencing, Staining, Transfection, Construct

Journal: The FEBS journal

Article Title: Y12C mutation disrupts IMPDH cytoophidia and alters cancer metabolism.

doi: 10.1111/febs.70086

Figure Lengend Snippet: Fig. 5. IMPDH2 Y12C point mutation lowers IMPDH levels in some cell types in vitro. (A) Western blot and quantification of IMPDH (normalised to ACTIN) in cultured HeLa, HEC-1-A, CE81T, and HCT116 cells. Lower IMPDH levels are observed in mutant HeLa and HCT116 cells. (B) Real-time PCR analysis of IMPDH1 and IMPDH2 mRNA levels in cultured cells. No significant difference in expression levels is observed in any of the mutant cell lines. Images are representative of n = 3 independent experiments performed in triplicate, and quantification data are obtained from the same experiments. Statistical significance is evaluated using Student’s t-test, with error bars representing SEM (**P < 0.01, ***P < 0.001).

Article Snippet: The following antibodies were used: rabbit anti-IMPDH2 polyclonal antibody (1 : 10 000, 12948-1-AP, ProteinTech), rabbit anti-HPRT polyclonal antibody (1 : 5000, GTX113466, GeneTex, Irvine, CA, USA), HRP-conjugated mouse anti-b-ACTIN monoclonal antibody (1 : 3000, HRP60008, ProteinTech) and HRP-conjugated goat anti-rabbit IgG polyclonal antibodies (1 : 10 000, 31460, Invitrogen).

Techniques: Mutagenesis, In Vitro, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction, Expressing

Journal: The FEBS journal

Article Title: Y12C mutation disrupts IMPDH cytoophidia and alters cancer metabolism.

doi: 10.1111/febs.70086

Figure Lengend Snippet: Fig. 6. IMPDH2 Y12C point mutation lowers IMPDH levels in some xenografts. (A) Western blot and quantification of IMPDH (normalised to ACTIN) in tumours derived from HeLa, HEC-1-A, CE81T, and HCT116 cells. A significant decrease in IMPDH levels is observed in mutant HEC-1-A and HCT116 xenografts. (B) Real-time PCR analysis of IMPDH1 and IMPDH2 mRNA levels in xenografts. Significant reductions in IMPDH1 or IMPDH2 expression levels are detected in some mutant tumours. Images are representative of n = 3 independent experiments performed in triplicate, and quantification data are obtained from the same experiments. Statistical significance is evaluated using Student’s t-test, with error bars representing SEM (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The following antibodies were used: rabbit anti-IMPDH2 polyclonal antibody (1 : 10 000, 12948-1-AP, ProteinTech), rabbit anti-HPRT polyclonal antibody (1 : 5000, GTX113466, GeneTex, Irvine, CA, USA), HRP-conjugated mouse anti-b-ACTIN monoclonal antibody (1 : 3000, HRP60008, ProteinTech) and HRP-conjugated goat anti-rabbit IgG polyclonal antibodies (1 : 10 000, 31460, Invitrogen).

Techniques: Mutagenesis, Western Blot, Derivative Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: The FEBS journal

Article Title: Y12C mutation disrupts IMPDH cytoophidia and alters cancer metabolism.

doi: 10.1111/febs.70086

Figure Lengend Snippet: Fig. 7. IMPDH2 Y12C point mutation alters the metabolic status of HeLa cell-derived xenografts. (A) Correlation heatmap of cultured HeLa cells and three pairs of HeLa cell-derived tumours (Ms 4, 5 and 6). (B) Metabolites discriminating between wild-type (WT) and mutant xenografts from Ms 4, 5, and 6, with variable importance in the projection (VIP) scores of 1.0 or higher. The bar on the right indicates whether each metabolite is more abundant (red) or less abundant (blue) in each group. (C, D) Nucleotide levels in three pairs of tumours (Ms 3, 13 and 15) derived from HeLa cells (C) and in cultured HeLa cells (D). Levels of GTP, UTP, and CTP are normalised to ATP levels.

Article Snippet: The following antibodies were used: rabbit anti-IMPDH2 polyclonal antibody (1 : 10 000, 12948-1-AP, ProteinTech), rabbit anti-HPRT polyclonal antibody (1 : 5000, GTX113466, GeneTex, Irvine, CA, USA), HRP-conjugated mouse anti-b-ACTIN monoclonal antibody (1 : 3000, HRP60008, ProteinTech) and HRP-conjugated goat anti-rabbit IgG polyclonal antibodies (1 : 10 000, 31460, Invitrogen).

Techniques: Mutagenesis, Derivative Assay, Cell Culture

Journal: The FEBS journal

Article Title: Y12C mutation disrupts IMPDH cytoophidia and alters cancer metabolism.

doi: 10.1111/febs.70086

Figure Lengend Snippet: Fig. 8. IMPDH2 Y12C point mutation alters metabolic flux in most tumours derived from HeLa, HEC-1-A and CE81T cells. Scatter plots showing the relative mRNA levels of selected genes involved in glycolysis, pentose phosphate pathway (PPP) and purine synthesis in mutant HeLa, HEC-1-A, CE81T, and HCT116 xenografts compared to their wild-type (WT) counterparts. Data are derived from n = 3 independent experiments performed in triplicate. Genes are colour-coded, with symbol shapes representing individual tumour samples. Grey areas indicate differences that are not statistically significant (Student’s t-test, P > 0.05).

Article Snippet: The following antibodies were used: rabbit anti-IMPDH2 polyclonal antibody (1 : 10 000, 12948-1-AP, ProteinTech), rabbit anti-HPRT polyclonal antibody (1 : 5000, GTX113466, GeneTex, Irvine, CA, USA), HRP-conjugated mouse anti-b-ACTIN monoclonal antibody (1 : 3000, HRP60008, ProteinTech) and HRP-conjugated goat anti-rabbit IgG polyclonal antibodies (1 : 10 000, 31460, Invitrogen).

Techniques: Mutagenesis, Derivative Assay