Journal: Scientific Reports

Article Title: Biallelic inheritance in a single Pakistani family with intellectual disability implicates new candidate gene RDH14

doi: 10.1038/s41598-021-02599-z

Figure Lengend Snippet: Western blotting showing GST pull-down of TMEM206/PACC1 from SK-N-SH cell lysate using wild-type (WT) versus mutant RDH14-GST constructs. A TMEM206 (PACC1) rabbit polyclonal antibody (1:1000 dilution; catalogue #TA342366; Origene Technologies, Rockville, MD) was used. ( A ) Input to lane 1 was SK-N-SH cell lysate (as a positive control). Lanes 2 and 3 show extracted protein for the RDH14-GST constructs, WT and mutant, respectively (as negative controls). In lanes 4, 5, and 6, the GST pull-down eluted protein, for WT and mutant RDH14-GST, and GST alone (as negative control), respectively (i.e. bait protein against prey input). ( B ) Flow-through from lanes 2–6 above was run and probed with the TMEM206 antibody. Western hybridization using GST antibodies was also performed to check sample loading (Supplementary Fig. ).

Article Snippet: The TMEM206/PACC1 protein was queried using anti-TMEM206 rabbit polyclonal antibody (1:1000 dilution; cat# TA342366; Origene Tech., Rockville, MD).

Techniques: Western Blot, Mutagenesis, Construct, Positive Control, Negative Control, Hybridization

Journal: Scientific Reports

Article Title: Biallelic inheritance in a single Pakistani family with intellectual disability implicates new candidate gene RDH14

doi: 10.1038/s41598-021-02599-z

Figure Lengend Snippet: Analysis of the RDH14 knock down effects on SOX2, MAP2, TMEM206 (PACC1) and BRINP2 (FAM5B) in SK-N-SH cells. ( A ) Western blot analysis of the RDH14-siRNA transfection showing knock down of RDH14 protein, using anti-RDH14 rabbit polyclonal antibody (1:1000 dilution; cat# HPA056686; Atlas Antibodies, Bromma, Sweden). ( B ) qRT-PCR showing effect of RDH14-siRNA on transcription of SOX2 in SK-N-SH cells. ( C ) Representative western blot images showing the effects of RDH14 siRNA knock down on SOX2 and MAP2 protein levels using anti-SOX2 monoclonal antibody (1:2000 dilution; cat# ab79351, Abcam Inc, Toronto, ON), and anti-MAP2 polyclonal antibody (1:2000 dilution; cat# ab79351, Abcam Inc, Toronto, ON). ( D ) Effect of RDH14 siRNA knock down on TMEM206/PACC1 and FAM5B/BRINP2 protein levels using anti-TMEM206 rabbit polyclonal antibody (1:1000 dilution; cat# TA342366; Origene Tech., Rockville, MD), and anti-FAM5B rabbit polyclonal antibody (1:500 dilution; cat# orb474203; Biorbyt Tech., St Louis, MO).

Article Snippet: The TMEM206/PACC1 protein was queried using anti-TMEM206 rabbit polyclonal antibody (1:1000 dilution; cat# TA342366; Origene Tech., Rockville, MD).

Techniques: Western Blot, Transfection, Quantitative RT-PCR

Journal: Genes

Article Title: Integration of Bioinformatics Resources Reveals the Therapeutic Benefits of Gemcitabine and Cell Cycle Intervention in SMAD4-Deleted Pancreatic Ductal Adenocarcinoma

doi: 10.3390/genes10100766

Figure Lengend Snippet: SMAD4 deletion correlated with an increase in gemcitabine sensitivity in PDAC. ( a ) The correlations between SMAD4 gene copy numbers (left part)/mRNA expression (right part) and gemcitabine drug activity were obtained from the CTRP database via an online tool, the CellMinerCDB; ( b ) PDAC cell lines were treated with gemcitabine for 72 h, and then cell viability was determined by an MTT assay. The SMAD4 mutation status was obtained from the CCLE database (missence mutation and frameshift insertion in AsPC-1, and HPAC cells, respectively). BxPC-3 cells harbored a homozygous deletion of SMAD4 gene . The values for SMAD4 copy number variation (CNV) and expression (EXP) were obtained from the CellMinerCDB database (CTRP-Broad-MIT data); ( c ) Chemosensitivity profiles for PDAC patient-derived organoids were obtained from a previous study. Values shown are the area under the curve (AUC) for each drug. Higher or lower AUC (area under the curve) indicated less or more responsive to each drug, respectively.

Article Snippet: AsPC-1, BxPC-3, HPAC, and PANC-1 human pancreatic cancer cells were purchased from the Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (Hsinchu, Taiwan).

Techniques: Expressing, Activity Assay, MTT Assay, Mutagenesis, Derivative Assay

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A. Schematic representation of the CAR construct used in this study: a second-generation CAR receptor containing a CD28 hinge and transmembrane domain, a CD28 costimulatory domain, and CD3ζ signaling domain. B. Schematic representation of the protocol used to manufacture CAR-T cells. Healthy donor-derived PBMCs were activated using an anti-CD3 antibody. After 48h, expanded T cells were transduced with a retroviral vector to induce expression of the CAR. Transduction efficiency was assessed 5-7 days after transduction and cells were used for functional analysis between 7-14 days after transduction. C. Schematic representation of the protocol used to detect phosphorylation in CAR-T cells. CAR-T cells were cocultured with PSCA-expressing HPAC (HPAC WT ) or PSCA-deficient HPAC (HPAC PSCA-KO ) tumor cells for 1, 10, 30 and 60 minutes, followed by protein extraction. For cytoplasmic proteins (ZAP70, PLCγ, VAV1), phosphorylation was detected in the whole cell extract (WCE). For detection of phosphorylated CD28 Y218, the CAR was enriched through immunoprecipitation. D. Left Panel. Representative Western blot showing the phosphorylation of CD28 Y218, Zap70 Y319, PLCγ Y783, VAV1 Y174 in PSCA-specific CAR-T cells, following stimulation with HPAC WT or HPAC PSCA-KO cells. Right Panel. Phosphorylation intensity quantified by densitometry (representative plot). CD28 pY218, ZAP70 pY319, PLCγ pY783 and VAV1 pY174 normalized with respect to total CAR (CD3ζ), total ZAP70, total PLCγ and total VAV1 respectively. Untransduced cells (UT) were included as a negative control of immunoprecipitation. E. T cells were isolated from PBMCs and stimulated using CD3/CD28 T -activator Dynabeads®. CD28 was then enriched through immunoprecipitation for further analysis. Left Panel. Representative Western blot showing the phosphorylation of endogenous CD28 Y218 at the indicated time points after stimulation. Right Panel. Phosphorylation intensity quantified by densitometry. CD28 pY218 was normalized with respect to total CD28.

Article Snippet: PSCA deficient HPAC cells (HPAC PSCA-KO ) were generated by Synthego (CA, USA) and a clonal population was derived by limiting dilution.

Techniques: Construct, Derivative Assay, Transduction, Retroviral, Plasmid Preparation, Expressing, Functional Assay, Phospho-proteomics, Protein Extraction, Immunoprecipitation, Western Blot, Negative Control, Isolation

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: Expression of PSCA in HPAC WT and HPAC PSCA-KO cells, measured by flow cytometry.

Article Snippet: PSCA deficient HPAC cells (HPAC PSCA-KO ) were generated by Synthego (CA, USA) and a clonal population was derived by limiting dilution.

Techniques: Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A . Schematic representation of second-generation chimeric antigen receptors targeting PSCA. The 218F mutant construct contains a point mutation in the AAYRS motif (the tyrosine (Y) is substituted with phenylalanine (F)). B . PSCA-targeted WT and 218F CAR-T cells were cocultured with HPAC WT cells at a 1:1 ratio. Supernatant was collected after 24 hours and cytokine production was measured by ELISA. Cytokine levels were normalized to that of WT CAR. Significance was determined by t-test with Welch’s correction. ** = P<0.01. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). C . Left panel. Schematic representation of the experimental design. CAR-T cells were cocultured with HPAC WT tumor cells at a 5:1 ratio. On days 2 and 4, 100.000 HPAC WT cells were added to the coculture and on day 7, cells were collected to be quantified. Right Panel. Number of CD3 + cells collected on day 7 after repetitive antigen stimulation. Significance was determined by paired t test. * = P<0.05. Each symbol represents an independent experiment from 4 different healthy donors. D. Left Panel. Schematic representation of the experimental design. NSG mice were injected subcutaneously with HPAC WT cells and 14 days after tumor injection, PSCA WT or 218F CAR-T cells were injected intravenously and tumor volume was measured for 35 days. Lower Left panel: CAR expression 1 day before adoptive cell transfer. Right panel: Tumor volume (mm 3 ) was monitored for 35 days (n=5). Representative example of two independent experiments. Significance was determined by linear regression and one-way ANOVA. * = P<0.05.

Article Snippet: PSCA deficient HPAC cells (HPAC PSCA-KO ) were generated by Synthego (CA, USA) and a clonal population was derived by limiting dilution.

Techniques: Mutagenesis, Construct, Enzyme-linked Immunosorbent Assay, Standard Deviation, Injection, Expressing

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A. Expression of CAR, CD4, and CD8 in WT and 218F CAR-T cells 7-10 days after transduction. Each point represents an individual healthy donor (n = 11). Statistical significance was determined by one-way ANOVA. B. The lysis of HPAC WT cells when treated with WT or 218F CAR-T cells was evaluated using a real-time cytotoxicity assay (xCELLigence). Left Panel: Representative graph showing % of cytolysis over a 72 hour period after the addition of the effector cells. Right Panel: % of cytolysis of WT and 218F CAR-T cells at 12 hours after the addition of the effector cells. Significance was determined by paired t-test. Each symbol represents an independent experiment using one of five different healthy donors. C. Proliferation capacity of WT vs 218F CAR-T cells, analyzed by CellTrace Violet dilution. WT and 218F CAR-T cells were co-cultured at a 2:1 E:T ratio and incubated at 37°C for 4 days. Percentage of Divided cells (left) and percentage of cells in the last division (right) of 218F CAR-T cells is shown normalized to WT CAR-T cells. Significance was determined by t-test with Welch’s correction. * = P<0.05. Each symbol represents an independent experiment from 4 different healthy donors. Data is represented as the mean ± standard deviation (SD). D. CD4 and CD8 CAR-T cells were sorted using magnetic MicroBeads. WT and 218F CAR-T cells were cocultured with HPAC WT cells at different CD4:CD8 ratios. Supernatant was collected after 24 hours and cytokine production was measured by ELISA. Left Panel. Representative graph showing the IL-2 production at different CD4:CD8 ratio. Dotted lines indicate IL-2 production by unsorted CAR-T cells. Center and left panel show production of IL-2 production of 218F CAR-T cells with respect to WT CAR-T cells. Significance was determined by t-test with Welch’s correction. ** = P<0.01. Each symbol represents an independent experiment from 3 different healthy donors.

Article Snippet: PSCA deficient HPAC cells (HPAC PSCA-KO ) were generated by Synthego (CA, USA) and a clonal population was derived by limiting dilution.

Techniques: Expressing, Transduction, Lysis, Cytotoxicity Assay, Cell Culture, Incubation, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: Transcriptomic profile of WT vs 218F CAR-T cells. CAR-T cells were cocultured with HPAC WT cells for 24 hours and subsequently sorted into CD4⁺ and CD8⁺ subsets using the MACSQuant® Tyto® system prior to RNA extraction. A. Principal component analysis (PCA) of WT and 218F CAR-T cells in all samples (left), CD4⁺ cells (center), and CD8⁺ cells (right). B. Gene expression of genes in the IL-17 signaling pathway. Enriched pathways between WT and 218F CAR-T cells were identified using IPA® software. Bar plots show log₂ fold change in gene expression in 218F CAR-T cells relative to WT CAR-T cells. CD4⁺ cells are shown on the left and CD8⁺ cells on the right.

Article Snippet: PSCA deficient HPAC cells (HPAC PSCA-KO ) were generated by Synthego (CA, USA) and a clonal population was derived by limiting dilution.

Techniques: RNA Extraction, Gene Expression, Software

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A-B . PSCA WT and 218F CAR-T cells were cocultured with HPAC WT cells for 0, 1, 10, 30 or, 60 min. A. After protein extraction, CAR-associated proteins were co-immunoprecipitated using protein-L coated beads. PLCγ abundance in the immunoprecipitate was analyzed by Western blot. Left panel: Representative Western blot. Right panel: Normalized CAR-bound PLCγ intensity quantified by densitometry (representative plot), and CAR-bound PLCγ 60 min post-stimulation, showing 3 biological replicates corresponding to different donors, analyzed in independent experiments. Densitometry values were normalized to total CAR (CD3ζ) B. SLP76 pY376 was analyzed using phospho-specific antibodies. Left panel: Representative Western blot of SLP76 pY376 in CAR-T cells after stimulation with HPAC WT cells. Right panel: Normalized SLP76 pY376 intensity quantified by densitometry (representative plot) and SLP76 pY376 at 60 min post-stimulation. Each symbol represents an independent experiment from 3 different healthy donors. Untransduced cells (UT) were included as a negative control of immunoprecipitation. Densitometry results were normalized relative to total SLP76. Significance was determined by paired t-test. * = P<0.05. C and E . Transcriptomic profile of WT vs 218F CAR-T cells. CAR-T cells were cocultured with HPAC WT cells for 24 hours and subsequently sorted into CD4⁺ and CD8⁺ subsets using the MACSQuant® Tyto® system prior to RNA extraction. C. Volcano plot representing the genes that are differentially expressed in 218F CAR-T cells vs WT CAR-T. CD4+ cell is the left, CD8+ cell in the right. D. CAR-T cells were cocultured with HPAC WT target cells and supernatant was collected 24hours hours after. IL-17A production was analyzed by ELLA. Cytokine production of 218F and UT was normalized to WT CAR. Significance was determined by t-test with Welch’s correction. * = P<0.05. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). E. Gene expression of cytokine-related genes associated with Th17 cells. Bar plot shows log₂ fold change in gene expression of 218F CAR-T cells relative to WT CAR-T cells. CD4⁺ cells are shown on the left and CD8⁺ cells on the right.

Article Snippet: PSCA deficient HPAC cells (HPAC PSCA-KO ) were generated by Synthego (CA, USA) and a clonal population was derived by limiting dilution.

Techniques: Protein Extraction, Immunoprecipitation, Western Blot, Negative Control, RNA Extraction, Standard Deviation, Gene Expression

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A . Top panel: Schematic representation of the protocol used to inhibit ITK activity. PSCA CAR-T cells were pre-treated for 2 hours with ITK inhibitors 1μM (BMS-509744, Ibrutinib or GNE-9822) or DMSO as control and then cocultured with HPAC WT cells for 0 or 10 minutes. Left panel: Representative Western blot of CD28 pY218 in CAR-T cells after stimulation with HPAC WT cells. Right panel: Normalized CD28 pY218 intensity with respect to CAR (CD3ζ). Significance was determined using one-way ANOVA. * = P<0.05. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). B. Top panel. Schematic representation of the experimental protocol. ITK expression was disrupted using CRISPR/Cas in Jurkat cells. WT and ITK-KO Jurkat cells were transduced to express a PSCA-specific CAR and cocultured with HPAC WT cells for 0 or 10 min. Left panel. Representative membrane of CD28 pY218 in CAR-T Jurkat cells after stimulation. Right panel. Normalized CD28 pY218 intensity quantified by densitometry (representative plot) and CD28 pY218 at 10 min post-stimulation. Significance was determined by paired t-test. ** = P<0.01. Each symbol represents one of 3 independent experiments, performed with different donor T cells.

Article Snippet: PSCA deficient HPAC cells (HPAC PSCA-KO ) were generated by Synthego (CA, USA) and a clonal population was derived by limiting dilution.

Techniques: Activity Assay, Control, Western Blot, Standard Deviation, Expressing, CRISPR, Membrane

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A . Schematic representation of WT and PYRP mutant CAR constructs. In the PYRP mutant, alanine (A) 217 and serine (S) 220 were substituted with prolines (P) to generate a proline-rich domain. B . PSCA-targeted WT and PYRP CAR-T cells were cocultured with HPAC WT cells for 0, 1, 10, 30, or 60 min. CAR molecules were immunoprecipitated using protein-L coated beads and co-immunoprecipitated proteins were analyzed by Western blot. Upper panel: Representative Western blot results showing ITK present in CAR immunoprecipitate. CD3ζ staining of the CAR molecule was used as a bait control. Lower panel: Intensity of ITK with respect a total CAR (CD3ζ) was analyzed by densitometry (representative plot). C. PSCA WT and PYRP CAR-T cells were stimulated with HPAC WT cells for 10 min and phosphorylation of Y218 was analyzed by mass spectrometry. Bar charts represent the intensity of phosphorylated Y218 normalized to the total CAR signal. Significance was determined by one-way Anova. * = P<0.05. Data is represented as the mean ± standard deviation (SD). D and F. PSCA WT and PYRP CAR-T cells were cocultured with HPAC WT cells. Supernatant was collected after 24 hours and cytokine production was measured by ELISA. Significance was determined by one-way ANOVA. * = P<0.05, ** = P<0.01. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). E. PSCA WT and PYRP CAR-T cells were cocultured with HPAC WT cells. RNA was extracted after 24 hours and IL-2 mRNA production was evaluated using quantitative Time PCR. CAR mRNA was used as normalization control. Significance was determined by one-way Anova. ** = P<0.01, *** = P<0.001. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). G. % of change in tumor volume (compared to baseline) is represented (n=5). Representative example of two independent experiments. Significance was determined by permutation two sample t-test. * = P<0.05.

Article Snippet: PSCA deficient HPAC cells (HPAC PSCA-KO ) were generated by Synthego (CA, USA) and a clonal population was derived by limiting dilution.

Techniques: Mutagenesis, Construct, Immunoprecipitation, Western Blot, Staining, Control, Phospho-proteomics, Mass Spectrometry, Standard Deviation, Enzyme-linked Immunosorbent Assay