rat igg2a isotype control antibody  (Bio X Cell)

Journal: Genes & Diseases

Article Title: Chloride channel accessory 4 suppresses stem cell-like properties of colorectal cancer and enhances anti-PD-1 immunotherapy

doi: 10.1016/j.gendis.2025.101859

Figure Lengend Snippet: CLCA4 suppressed colorectal cancer stem cell expansion by interacting with vimentin to suppress FAK signaling pathways. (A) Western blotting analysis of FAK and p-FAK protein levels in control and CLCA4-overexpressing colorectal cancer (CRC) cells. Right panels: Quantification of protein expression ratio. (B) Western blotting analysis of stemness-related proteins and p-FAK in CLCA4-overexpressing cells treated with or without FAK agonist. Lower panels: Quantification of protein expression ratio. (C) Tumorsphere formation assay was performed to examine the tumorsphere formation ability in CLCA4-overexpressing cells treated with or without FAK agonist. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation). (D) Immunoprecipitation and IgG samples were analyzed by mass spectrometry. Proteins with unused >1.3 were filtered out, and keratin was removed. A total of 336 proteins were identified, including 334 proteins in immunoprecipitation samples and 4 proteins in IgG samples. (E) The immunoprecipitates of CLCA4 were purified using anti-Flag antibody and separated with SDS-PAGE, and the presence of vimentin was analyzed by Western blotting. Normal IgG was used as the negative control. (F) The immunoprecipitates of vimentin were purified using anti-HA antibody and separated with SDS-PAGE, and the presence of CLCA4 was analyzed by Western blotting. Normal IgG was used as the negative control. (G) The differences in protein levels (vimentin, Bmi-1, and p-FAK) among CRC cells transfected with different plasmids were analyzed by Western blotting. Right panels: Quantification of protein expression ratio. (H) Tumorsphere formation assay was performed to examine the tumorsphere formation ability among CRC cells transfected with different plasmids. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation).

Article Snippet: After 7 days, mice were intraperitoneally treated with either an in vivo blocking antibody against mouse PD-1 (Clone: 29F.1A2, BioXcell, Cat# BP0273) or a rat IgG2a isotype control antibody (Clone: 2A3, BioXcell, Cat# BP0089).

Techniques: Protein-Protein interactions, Western Blot, Control, Expressing, Tube Formation Assay, Standard Deviation, Immunoprecipitation, Mass Spectrometry, Purification, SDS Page, Negative Control, Transfection

Journal: bioRxiv

Article Title: Arginine synthesis pathway and ASS1 play a critical role in mRNA translation reprogramming and ICI resistance in cutaneous melanoma

doi: 10.64898/2026.03.17.712479

Figure Lengend Snippet: A , C57/BL6 mice were inoculated with murine Braf V600E Pten -/- melanoma cells (YUMM1.1), expressing a shRNA targeting ASS1 or a shRNA control. When tumors reached a mean of 100mm3, mice were treated with anti-PD-1 (4mg/kg-1) or isotype IgG control. The data are presented as the mean ± SEM for each group (n=10 mice per group). p-values were calculated using two-way ANOVA. B , Percentage of CD45-positive cells in tumors was determined by flow cytometry in tumors presented in B. The data are presented as the mean ± SEM (YUMM1.1shCTL n=9 tumors ; YUMM1.1shASS1 n=10 tumors). C , Deconvolution performed using CIBERSORTx performed on tumors from the experiment presented in A. D , Ratio CD8+ cells to malignant cells (Mal.) from the panel C is presented in tumor from the experiment presented in the panel A. E , Representative immunostaining of CD8+ T lymphocytes (red, CD8a) and nuclei (blue, DAPI) on frozen tumor sections of mice from panel A. F, Representation of CD8+ cells shown in D, the data are presented as the mean ± SEM (n=9 tumors). G. Representative immunostaining of CD8+ T cells and granzyme B on frozen tumor sections of mice from panel A.

Article Snippet: Antibodies InVivo Mab anti-mouse PD-1 (CD279), clone 29F.1A12 or InVivo Mab rat IgG2a isotype control, clone 2A3 were purchased from BioXCell, diluted in PBS and injected intraperitoneally (100μg in 100μL of PBS; 4mg.kg -1 per mouse).

Techniques: Expressing, shRNA, Control, Flow Cytometry, Immunostaining

Journal: bioRxiv

Article Title: Arginine synthesis pathway and ASS1 play a critical role in mRNA translation reprogramming and ICI resistance in cutaneous melanoma

doi: 10.64898/2026.03.17.712479

Figure Lengend Snippet: A , Immunoblot showing the expression levels of indicated protein in ICI-resistant YUMM1.1 cell line after 3 days of treatment with MDLA. HSP90 serves as a loading control. B , ASS1 enzymatic activity was performed in ICI-resistant YUMM1.1 cell line after 3 days of treatment with (n=3; mean ± SEM). C , Intracellular concentration of arginine was measure in ICI-resistant YUMM1.1 cells after 3 days of treatment with MDLA. (n=3; mean ± SEM). D. Schematic representation of the impact of arginine on the control upstream of mTORC1. E. Immunoprecipaption was performed on WM9 cells transfected with HA-tagged CASTOR1 and cultured for 3 days in the presence of different concentration of arginine and MDLA. Immunoblot showing the expression levels of indicated protein. F , Immunoblot showing the expression levels of indicated protein in ICI-resistant YUMM1.1 cell line. β-actine was use as a loading control. G , Immunoblot showing the expression levels of indicated protein in WM9 melanoma cell line. H , Renilla over Firefly luminescent ratio quantification in ICI-resistant YUMM1.1 cell line. I , Renilla over Firefly luminescent ratio quantification in WM9 melanoma cell line. J , YUMM1.1 cells treated or not with MDLA were then pulsed for 30 min with [ 35 S]Cys/Met, and the incorporation of 35 S into proteins was quantified and normalized to the total protein amount. Cells treated 30 minutes with 100µg/mL of cycloheximide (CHX) are used as a positive control. The data are presented as the mean (n = 3). K , WM9 cells treated or not with MDLA were then pulsed for 30 min with [ 35 S]Cys/Met, and the incorporation of 35 S into proteins was quantified and normalized to the total protein amount. Cells treated 30 minutes with 100µg/mL of cycloheximide (CHX) are used as a positive control. The data are presented as the mean (n = 3). L , Immunoblot showing the expression levels of indicated protein in cells from tumors of patients with metastatic melanoma. HSP90 serves as a loading control. M , Renilla over Firefly luminescent ratio quantification in cells from tumors of patients with metastatic melanoma (n=3; mean ± SEM). N , Cells isolated from tumors of patients with metastatic melanoma were treated or not with MDLA were then pulsed for 30 min with [ 35 S]Cys/Met, and the incorporation of 35 S into proteins was quantified and normalized to the total protein amount. Cells treated 30 minutes with 100µg/mL of cycloheximide (CHX) are used as a positive control. The data are presented as the mean (n = 3). O , C57BL/6 mice were inoculated with murine Braf V600E Pten -/- melanoma cells (YUMM1.1). When tumors reached a mean of 100mm 3 , mice were treated with MDLA (700mg.kg-1) or PBS, twice a day. Two days after the beginning of the MDLA treatment, mice were treated with anti-PD-1 (4mg/kg-1) or isotype IgG control, once per day, every two days, 3 times. Mice were treated with an additional dose of anti-PD-1 or isotype IgG control at day 24. Tumor growth was monitored for 27 days. The data representing the tumor growth are presented as the mean ± SEM for each group (n=10 mice per group). p -values were calculated using two-way ANOVA. P , Percentage of CD45-positive cells in tumors was determined by flow cytometry in tumors presented in P. The data are presented as a ratio of percentage between CD45-positive cells versus CD45-negative cells. (n=10 tumors per group, mean ± SEM). Q , Percentage of CD45 and CD8a-double positive cells in tumors, determined by flow cytometry in tumors presented in P. (n=10 tumors per group, mean ± SEM).

Article Snippet: Antibodies InVivo Mab anti-mouse PD-1 (CD279), clone 29F.1A12 or InVivo Mab rat IgG2a isotype control, clone 2A3 were purchased from BioXCell, diluted in PBS and injected intraperitoneally (100μg in 100μL of PBS; 4mg.kg -1 per mouse).

Techniques: Western Blot, Expressing, Control, Activity Assay, Concentration Assay, Transfection, Cell Culture, Positive Control, Isolation, Flow Cytometry