ly 6g magnetic bead pulldown  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Microglial lipid signaling drives glioblastoma invasion and represents a therapeutic vulnerability

doi: 10.64898/2026.04.24.720633

Figure Lengend Snippet: (A) Gene expression of Yap-target genes ( Ankrd1, Ctgf, Cyr61 ) in NSCG (left) and NFpp10 (right) cells upon 24 hours of treatment with BV2 CM, BV2 CM LRA (lipid removal), BV2 CM LRA (lipid removal) LPA 18:1. Relative gene expression to S18. Fold increase. NSCG: n=3, 2way ANOVA, ** p-value<0.0056; *** p-value<0.0003, **** p-value<0.0001; NFpp10: n=3, 2way ANOVA, * p-value<0.0381, ** p-value=0.0012, *** p-value=0.0002, **** p-value<0.0001. (B) Representative immunoblots of Yap nuclear and cytoplasmic levels in NFpp10 cells upon 5 minutes, 10 minutes, 20 minutes of co-culture with BV2 cells and LPA 16:0 treatment. (C) Representative immunoblots of RhoA pulldown and total in NSCG (left) and NFpp10 (right) cells upon 2 minutes, 5 minutes, 10 minutes co-culture with BV2 cells. (D) Representative immunoblots of RhoA pulldown and total in NSCG (left) and NFpp10 (right) cells upon 5 minutes, 10 minutes co-culture with BV2 cells and LPA 18:1 or LPA 16:0 treatment respectively. (E) Invasion assays of NSCG (left) and NFpp10 (right) Scr (control) and YAP/TAZ knock-out cells in the presence or absence of BV2 cells. Fold increase. NSCG: n=3, One-way ANOVA, * p-value=0.0267, *** p-value=0.0001; NFpp10: n=6, One-way ANOVA, * p-value=0.0432, ** p-value=0.0034. (F) Invasion assays of NSCG Scr (control) and YAP knock-out cells in the presence or absence of BV2 cells and LPA 18:1 treatment. Fold increase. NSCG: n=3, One-way ANOVA, ** p-value<0.0022, *** p-value=0.0004, **** p-value<0.0001. (G) Tumor cell number across the 50 distinct TMAs. h. Ratio tumor cell/microglia-macrophages across the selected 15 tumor core and 15 tumor rim TMAs based on tumor cell density. i. Correlation tumor cells/microglia-macrophages across the selected 15 tumor core and 15 tumor rim TMAs. Each dot/number refers to a TMA sample. Correlation index: −0.477. All data represent mean ± SEM.

Article Snippet: Protein extraction was performed using the RhoA Pulldown activation Assay Kit (Cytoskeleton, BK036).

Techniques: Gene Expression, Western Blot, Co-Culture Assay, Control, Knock-Out

Journal: bioRxiv

Article Title: A class act: HDAC1- Malat1 regulates MDSC apoptosis and cell cycling to decrease suppression of T cells

doi: 10.64898/2026.03.23.713743

Figure Lengend Snippet: (A) Western blots of Tyr705 phosphorylated-STAT3 (pSTAT3), STAT3, and Beta-actin in G-MDSCs isolated from NT2.5-LM metastatic lung tumors. G-MDSCs were isolated with Ly-6G+ magnetic beads from single cell suspensions of dissociated metastatic lung tumors and stimulated with LPS (2 hours, 1 ug/mL). NT2.5-LM tumor-bearing mice were treated with vehicle vs. Entinostat (5 mg/kg, 5x/wk) for 3 weeks. Each lane represents one mouse (n=5 per treatment group). 50 ug of total protein lysate loaded in each lane. Band density quantified with Image J and adjusted density normalized to Beta-actin (right), outlier mouse #2 in Vehicle removed with ROUT at threshold Q=1%. Two-way ANOVA, * p<0.05. (B) Western blot of pSTAT3, STAT3, and Beta-actin in J774M cells after 24 hours of Entinostat treatment and IFN-γ stimulation (30 min, 20 ng/mL). 20 ug of total protein lysate loaded in each lane. (C) Western blot of pSTAT3, STAT3, and Beta-actin in hMDSCs after 24 hours of Entinostat treatment. 50 ug of total protein lysate loaded in each lane. (D) Representative flow plots (left) and quantification (right, n=3) of J774M STAT3-responsive GFP reporter after 18 hours of pre-treatment with various concentrations of various HDAC inhibitors and IFN-γ stimulation for 5-7 hours (20 ng/mL), indicative of percentage of STAT3 activation. One-way ANOVA for (D: all statistically significant with p<0.05, unless indicated as non-significant (ns)). ENT = Entinostat; PAN = Panobinostat; BEL = Belinostat; VOR = Vorinostat; TSA = Trichostatin A; DOM = Domatinostat; CHL = Chlopynostat; PYR = Pyroxamide; SCA = Santacruzamate A; RGF = RGFP966; TMP = TMP269; SIS = SIS17.

Article Snippet: G-MDSCs were isolated from single-cell suspensions of NT2.5-LM lung metastases after 3 weeks of vehicle vs. Entinostat treatment (as described above) using a Ly-6G+ magnetic bead pulldown from the MDSC isolation kit (Miltenyi, cat. #130-094-538), following manufacturer’s instruction.

Techniques: Western Blot, Isolation, Magnetic Beads, Single Cell, Activation Assay