rabbit anti npepps  (Boster Bio)

Journal: bioRxiv

Article Title: NPEPPS regulates intracellular import and sensitivity to cisplatin by interaction with volume regulated anion channels

doi: 10.1101/2021.03.04.433676

Figure Lengend Snippet: ( A ) Differential gene expression of the 46 common synthetic lethal genes as measured by RNAseq across all cell lines, comparing the treatment resistant derivative (Gem-, Cis-, GemCis-resistant) to the associated parental cell line. Asterisks indicate a statistically significant result (moderated t-test, *FDR < 0.05). The bar plot on top is the aggregate count of significant results across all 15 comparisons. Genes are ranked by the count of statistically significant upregulated hits. ( B ) RNAseq (compared to parentals; *FDR < 0.05), ( C ) mass spectrometry proteomics (compared to parentals, *FDR < 0.25), and ( D ) CRISPR screen results for NEPPSP (*FDR < 0.05). ( E ) Representative immunoblots and densitometry quantification for independent triplicates (mean ± SEM) for NPEPPS in all cell lines (*FDR < 0.05).

Article Snippet: NPEPPS and LRRC8A has been probed using Rabbit polyclonal NPEPPS antibody (1:1000; Origene), Rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio) and Rabbit IgG polyclonal LRRC8D antibody (1:1000, SinoBiological).

Techniques: Gene Expression, Mass Spectrometry, CRISPR, Western Blot

Journal: bioRxiv

Article Title: NPEPPS regulates intracellular import and sensitivity to cisplatin by interaction with volume regulated anion channels

doi: 10.1101/2021.03.04.433676

Figure Lengend Snippet: ( A ) NPEPPS was found to be synthetic lethal with cisplatin in a CRISPR screen for 27 genotoxic agents in RPE1 cells by . ( B ) Immunoblot for NPEPPS across several control and shRNAs targeting NPEPPS. ( C, D ) KU1919-GemCis cells with knockdown of NPEPPS treated with increasing doses of cisplatin or gemcitabine. A total of 3 technical replicates per dose (mean ± SEM). Independent experiments are reported in Figure S5 . ( E ) NPEPPS mRNA is upregulated in response to cisplatin treatment in a dose dependent manner in both KU1919-Par and KU1919-GemCis cells. Independent triplicate experiments are shown (mean ± SEM) (t-test compared to 0μM; *p < 0.05, **p < 0.05). ( F ) Pharmacologic targeting of NPEPPS with tosedostat in GemCis-resistant cells treated with cisplatin, gemcitabine, or gemcitabine plus cisplatin. A total of 3 technical replicates per dose are shown (mean ± SEM). Independent experiments are reported in Figure S6 .

Article Snippet: NPEPPS and LRRC8A has been probed using Rabbit polyclonal NPEPPS antibody (1:1000; Origene), Rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio) and Rabbit IgG polyclonal LRRC8D antibody (1:1000, SinoBiological).

Techniques: CRISPR, Western Blot, Control, Knockdown

Journal: bioRxiv

Article Title: NPEPPS regulates intracellular import and sensitivity to cisplatin by interaction with volume regulated anion channels

doi: 10.1101/2021.03.04.433676

Figure Lengend Snippet: ( A ) NPEPPS is found to interact with all VRAC subunits, LRRC8A-E, as reported in the BioPlex interactome . ( B ) Anti-FLAG was used against KU1919 and T24 parental cell lines as controls and overexpressing FLAG tagged NPEPPS, KU1919 and T24 cells. The immunoprecipitant was immunoblotted for NPEPPS, LRRC8A, and LRRC8D, demonstrating that LRRC8A and LRRC8D are pulled down in complex with NPEPPS. ( C ) Genes ranked based on log2 fold change from the synthetic lethal CRISPR screens across all cell lines. LRRC8A-E and the 46 common synthetic lethal genes are labeled. ( D, E ) Knockout of LRRC8A and LRRC8D through the CRISPR screen resulted in increased cell growth upon gemcitabine plus cisplatin treatment in GemCis- resistant cell lines (moderated t-test; *FDR < 0.05). ( F, G ) LRRC8A and LRRC8D gene expression measured by RNAseq (compared to parentals; *FDR < 0.05).

Article Snippet: NPEPPS and LRRC8A has been probed using Rabbit polyclonal NPEPPS antibody (1:1000; Origene), Rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio) and Rabbit IgG polyclonal LRRC8D antibody (1:1000, SinoBiological).

Techniques: CRISPR, Labeling, Knock-Out, Gene Expression

Journal: bioRxiv

Article Title: NPEPPS regulates intracellular import and sensitivity to cisplatin by interaction with volume regulated anion channels

doi: 10.1101/2021.03.04.433676

Figure Lengend Snippet: ( A ) Volcano plot of metabolites measured from KU1919-GemCis cells with or without NPEPPS knockdown (shN39). Time and treatment (cisplatin 10μM) were covariates in the linear model to calculate differential expression using a moderated t-test; horizontal grey line is -log10(FDR = 0.05). ( B )Taurine abundance measured in KU1919-GemCis cells with non- targeting shRNA controls or shRNA targeting NPEPPS (shN39). Cells were also measured at 48 hours treated with 10μM cisplatin or PBS. ( C ) Intracellular cisplatin levels in ( C ) KU1919 or ( D ) 5637 cells were measured after 4 hours of 10μM cisplatin treatment using CyTOF, with the number of cells analyzed as indicated. Immunoblot of LRRC8A, LRRC8D, and γH2AX in ( E ) KU1919-GemCis-shCtrl1 and KU1919- GemCis-shN39 or ( F ) 5637-GemCis-shCtrl1 and 5637-GemCis-shN39 cells treated with PBS or 10μM cisplatin for 48 hours. ( G ) Intracellular cisplatin concentrations were measured for KU1919 parental and then for untargeted knockdown (siCtrl) and targeted knockdown of NPEPPS (siNPEPPS), LRRC8A (siLRRC8A), and the combination of NPEPPS and LRRC8A (siNPEPPS+siLRRC8A). ( H ) Tumor volume of KU1919-GemCis xenografts measured over time and across 4 treatment groups considering non- targeting shRNA controls (shCtrl1), shRNA targeting NPEPPS (shN39), PBS vehicle control (PBS), or gemcitabine plus cisplatin treatment (GemCis). ( I ) Survival analysis of xenograft models with a defined endpoint of a tumor volume > 2cm 3 . Logrank test was applied to test significance. ( J ) Survival analysis of muscle-invasive bladder cancer in the TCGA stratified based on copy number amplification, gain or overexpression of LRRC8A or LRRC8D. Patients all had a record of cisplatin-based chemotherapy treatment. ( K ) Survival analysis for patients stratified by LRRC8A or LRRC8D as in ( J ), but that did not have any record of cisplatin-based treatments.

Article Snippet: NPEPPS and LRRC8A has been probed using Rabbit polyclonal NPEPPS antibody (1:1000; Origene), Rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio) and Rabbit IgG polyclonal LRRC8D antibody (1:1000, SinoBiological).

Techniques: Knockdown, Quantitative Proteomics, shRNA, Western Blot, Control, Amplification, Over Expression

Journal: bioRxiv

Article Title: NPEPPS regulates intracellular import and sensitivity to cisplatin by interaction with volume regulated anion channels

doi: 10.1101/2021.03.04.433676

Figure Lengend Snippet: Normal functioning cells will import cisplatin through the volume regulated anion channels (VRAC), with LRRC8A and LRRC8D being the primary subunits. A mechanism of cisplatin resistance is to inherently down-regulate VRACs. We propose that NPEPPS interacts with LRRC8A or LRRC8D directly to decrease VRAC activity, which prevents export of taurine and import of cisplatin, hence driving cisplatin resistance.

Article Snippet: NPEPPS and LRRC8A has been probed using Rabbit polyclonal NPEPPS antibody (1:1000; Origene), Rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio) and Rabbit IgG polyclonal LRRC8D antibody (1:1000, SinoBiological).

Techniques: Activity Assay