Journal: The EMBO Journal

Article Title: Micropeptide hSPAR regulates glutamine levels and suppresses mammary tumor growth via a TRIM21-P27KIP1-mTOR axis

doi: 10.1038/s44318-024-00359-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The membranes were blocked in 5% BSA (Sangon, China) for 1 h at room temperature, and then incubated at 4 °C overnight with primary antibody GAPDH (Proteintech, USA, 60004-1-Ig), hSPAR (HuaBio, China), Flag (Abcam, UK, ab205606), β-Tubulin (Proteintech, USA, 10068-1-AP), FIBRILLARIN (Proteintech, USA, 16021-1-AP), ATPV1A (Proteintech, USA, 14418-1-AP), LAMP2 (CST, USA, 49067), TRIM21 (Proteintech, USA, 67136-1-Ig), P27KIP1 (Proteintech, USA, 25614-1-AP), phospho-P27KIP1 (Abcam, USA, ab75908), SKP2 (Proteintech, USA, 15010-1-AP), SLC7A1 (Proteintech, USA, 14195-1-AP), SLC7A5 (Proteintech, USA, 28670-1-AP), phospho-mTOR (CST, USA, 5536), mTOR (CST, USA, 2983), phospho-S6K (CST, USA, 9234), S6K (CST, USA, 2708), phospho-S6 (CST, USA, 2211), S6 (CST, USA, 2217), phospho-AKT (CST, USA, 4060), AKT (CST, USA, 9272), Ubiquitin (Abcam, UK, ab134953), SLC38A2 (ImmunoWay, USA, YT4354), LAMTOR1(CST, USA, 8975), LAMTOR2 (CST, USA, 8145), LAMTOR3 (Proteintech, USA, 14492-1-AP), LAMTOR4 (CST, USA, 13140), LAMTOR5 (Proteintech, USA,11937-1-AP), RagA (CST, USA, 4357S) and TAT (Abcam, UK, ab42359).

Techniques: Recombinant, Sequencing, Modification, Membrane, Lysis, Transfection, Magnetic Beads, Software, Cytometry, In Vitro, cDNA Synthesis, Plasmid Preparation, Isolation, Mutagenesis, Silver Staining

Journal: bioRxiv

Article Title: Metabolic immunity to infection is driven by mitochondrial one-carbon metabolism

doi: 10.1101/2025.05.07.652698

Figure Lengend Snippet: ( A ) Immunoblot (IB) analyses of lysates from WT and eIF2α S51A AN3-12 mouse embryonic stem cells that were either uninfected, infected with Toxoplasma , or tunicamycin-treated (3 μg/ml) for 24 hours: ATF4, ∼50 kDa; phospho-EIF2α (Ser51), ∼38 kDa; α-Tubulin (TUBA), ∼55 kDa; and Toxoplasma GAP45 (TgGAP45), ∼45 kDa. ( B ) Heatmap of mRNA levels of ATF4 target genes in uninfected, infected and tunicamycin (3 μg/ml) treated ES-2 cells; n=3 independent cultures; SHMT2 and MTHFD2 are boxed. ( C and D ) WT and ATF4 KO ES-2 cells were uninfected or infected with Toxoplasma for 24 h and analyzed by qPCR for SHMT2 and MTHFD2 . Transcript levels were normalized to ACTB and are relative to WT uninfected. Data are mean ± SD of n=7 independent cultures. ****p<0.0001 for uninfected versus infected by means of two-way ANOVA analysis. ( E ) IB of ES-2 cells of the indicated genotype: MTHFD2, ∼32 kDa; α-Tubulin (TUBA), ∼55 kDa. ( F, G and H ) ES-2 cells of the indicated genotype were analyzed for mtDNA by qPCR for DLOOP , mt -ND1 and mt -CTYB normalized to RUNX2 levels and relative to WT uninfected. Data are mean ± SD of n=4 independent cultures. *p<0.05; for uninfected versus Toxoplasma infected, and #p<0.05; ##p<0.01 for WT versus MTHFD2 KO by means of two-way ANOVA analysis. ( I ) IB analysis of lysates from ES-2 cells that were uninfected or infected with indicated Toxo strains (MOI: 4) at 24 hpi: ATF4, ∼50 kDa; Vinculin (VCL), ∼124 kDa; and Toxoplasma GAP45 (TgGAP45), ∼45 kDa. ( J and K) Cells treated as in ( I ) were analyzed by qPCR for MTHFD2 and ASNS . Transcript levels were normalized to ACTB levels and are relative to uninfected cells. Data are mean ± SD of n=4 independent cultures, *p<0.05; **p<0.01 by means of one-way ANOVA analysis. ( L ) WT and ATF4 KO ES-2 were infected with Toxoplasma ± ISRIB (200nM) and analyzed 24 hpi by means of flow cytometry for Toxoplasma burden (mCherry median FI). Data are mean ± SEM of n=3 biological experiments, **p< 0.01 for DMSO versus ISRIB, #### p < 0.0001 for WT versus ATF4 KO by means of two-way ANOVA analysis. ( B - L ) ES-2 cells used for all experiments.

Article Snippet: The following antibodies were used: ATF4 (CST #11815), VCL (CST #4650), pS6K (CST #97596), TUBA (Proteintech 66031-1-Ig), MTHFD2 (CST #41377), SHMT1 (CST #80715), MTHFD1L (Invitrogen #PA5-100158), MTHFD1 ( Sigma #HPA015006), phospho-eIF2a (Ser51) (CST #9721), ACTB (CST #4970), TgGra45 (Dr. D Soldati; U. of Geneva) and TgGRA7 (Dr. J Boothroyd; Stanford University).

Techniques: Western Blot, Infection, Flow Cytometry