Journal: Cell Reports Methods

Article Title: MPA PASS software enables stitched multiplex, multidimensional EV repertoire analysis and a standard framework for reporting bead-based assays

doi: 10.1016/j.crmeth.2021.100136

Figure Lengend Snippet: Detection antibodies used

Article Snippet: CA9 , Miltenyi Biotec , Cat# 130-110-058; RRID: AB_2651327.

Techniques: Labeling, Recombinant

Journal: Cell Reports Methods

Article Title: MPA PASS software enables stitched multiplex, multidimensional EV repertoire analysis and a standard framework for reporting bead-based assays

doi: 10.1016/j.crmeth.2021.100136

Figure Lengend Snippet:

Article Snippet: CA9 , Miltenyi Biotec , Cat# 130-110-058; RRID: AB_2651327.

Techniques: Clinical Proteomics, Recombinant, Saline, Modification, Software

Journal: bioRxiv

Article Title: Chromosome-Specific Aneuploidy Engineering via dCas9-Induced Centromeric Chromatin Relaxation

doi: 10.1101/2025.04.25.650684

Figure Lengend Snippet: a , Schematics showing the experiment setup of Chr3 aneuploidy engineering using CRISPRt with Chr3-sg2 (Chr3-Ct) or NT-sg1 (CTRL), and VHL allele-specific KO ( VHL -ASK) in K1088N PTECs. Cells were cultured for 7 days (D7) or 11 days (D7+4) after treatments. Cells with Chr3-Ct or VHL -ASK on D7+4 were selected for CA9-positive (CA9+) cells with flow sorting. The six samples fixed for scRNA-seq are marked in red. b, Heatmaps showing single-cell chromosome copy number profiles inferred from scRNA-seq data of the indicated samples. The number of cells analysed (n) is shown. Rows represent individual cells and columns represent chromosomes. Copy number status is shown in blue (loss), red (gain) or white (neutral). c, Violin plots showing InferCNV residual expression for Chr3p and Chr3q of the indicated samples. The dots represent individual cells. The blue and red dashed lines represent the thresholds (see Methods) for copy number loss and gain, respectively. The cell proportions with the indicated copy number status are shown in the tables.

Article Snippet: Cells were stained with 1:50 diluted anti-CA9-PE antibody (Miltenyi, 130-131-321) in flow sorting buffer (1% bovine serum albumin (BSA) and 1μM EDTA in PBS) for 30 min at 4 °C in the dark.

Techniques: Cell Culture, Expressing

Journal: bioRxiv

Article Title: Chromosome-Specific Aneuploidy Engineering via dCas9-Induced Centromeric Chromatin Relaxation

doi: 10.1101/2025.04.25.650684

Figure Lengend Snippet: a , Schematics showing the expected genotypes generated after Chr3-Ct or VHL -ASK in K1088N PTECs with the corresponding VHL status annotated ( VHL + for VHL proficient, and VHL- for VHL loss) and the target genotypes with VHL loss enriched by CA9-positive (CA9+) flow sorting. b, Scatter plots showing the CA9 and DAPI staining intensities of K1088N PTECs after the indicated treatment. The rectangular box shows the gating strategy to flow sort for live (DAPI-) CA9-positive (CA9+) cells. c, Violin plots showing the signature scores of a curated HIF pathway activation gene set (see Methods) in K1088N PTECs after the indicated treatment. The dots represent individual cells. The dashed line represents the calculated threshold (see Methods) for defining cells with a high (HIF-High) or a low (HIF-Low) HIF pathway signature score, used for assigning VHL proficient ( VHL +) or VHL loss ( VHL- ) status.

Article Snippet: Cells were stained with 1:50 diluted anti-CA9-PE antibody (Miltenyi, 130-131-321) in flow sorting buffer (1% bovine serum albumin (BSA) and 1μM EDTA in PBS) for 30 min at 4 °C in the dark.

Techniques: Generated, Staining, Activation Assay

Journal: bioRxiv

Article Title: Cytoplasmic mRNA capping enzyme is regulated by hypoxia inducible factor HIF1α and controls stability of the target lncRNAs during hypoxia

doi: 10.1101/2024.07.05.602207

Figure Lengend Snippet: A. Biochemical fractionation of normoxic/hypoxic U2OS cells into nuclear/ cytoplasmic fractions. The quality of fractionation was assessed by immunoblotting of nuclear marker Lamin A/C) and cytoplasmic marker (GAPDH). CA9 immunoblotting was done to confirm the hypoxic condition. B. Realtime PCR of cCE-targeted lncRNAs shows differential expression patterns in cytoplasmic extracts of hypoxic osteosarcoma cells. Hypoxia significantly elevates the expression of ASH1L-AS1, GAS5-215, ENTPD3-AS1, and H19-009, whereas NFYC-AS1 expression was significantly reduced. Two-tail Student’s t-test was performed for statistical analysis C. Schematic representation of the dominant negative form of cytoplasmic restricted capping inhibited construct (K294A) BIO and myc denote the biotinylation and myc tag respectively. The nuclear export sequence (NES) of HIV is inserted in the N-terminus and the Nuclear Localization Signal (NLS) from the C-terminus is deleted to restrict expression of CE only in the cytoplasm. The lysine (K) residue at 294 th position is mutated to alanine (A) to make the construct catalytically inactive. D. Immunofluorescence image of myc tagged K294A expressing cells. E. Nuclear and cytoplasmic fractionation of U2OS cells expressing ±K294A under normoxic/hypoxic conditions. Western blot of Myc and CA9 confirm K294A expression and hypoxic condition respectively. The quality of fractionation was further substantiated by western blotting of nuclear marker (Lamin A/C) and cytoplasmic marker (GAPDH). F. The steady- state expression of GAS5-215, NFYC-AS1, and H19-009 was significantly decreased in K294A- expressing cells under normoxia. But ASH1L-AS1 was significantly increased and ENTPD3-AS1 did not show any significant change. G. The steady State expression of lncRNAs in hypoxic K294A cells. Only H19-009 showed significantly reduced expression in hypoxic K294A cells. Statistical significance was calculated by Two-tail Student’s t-test. Values are represented as ± SD from three biological replicates. ns, P: non-significant, *P< 0.05, **P< 0.005, ***P< 0.0005, ****P< 0.0001; n≥3.

Article Snippet: The dilutions are as follows 1:1000 rabbit anti-HIF1α (Cell Signaling Technology, cat no# 36169); 1:1000 mouse anti-beta actin (Santa Cruz Biotechnology, cat no# SC-517582), 1:1000 rabbit anti-CE (Novus Biologicals, cat no# NPB1- 49973); 1:2000 mouse anti-GAPDH (Novus Biologicals, cat#2D4A7); 1:2000 rabbit anti-CA9 (Cloud-clone Crop, cat# PAD076Hu01); 1:2000 mouse anti-lamin (DHSB, cat no# MANLAC1(4A7) at 4 JC overnight with constant shaking.

Techniques: Fractionation, Western Blot, Marker, Expressing, Dominant Negative Mutation, Construct, Sequencing, Residue, Immunofluorescence