Journal: bioRxiv

Article Title: Insights into the regulation of VPS13 family bridge-like lipid transfer proteins from the structure of VPS13C

doi: 10.1101/2025.11.10.687702

Figure Lengend Snippet: (A) Predicted interaction of Saccharomyces cerevisiae ( Sc ) VPS13 (light blue) with CaM (yellow) through a conserved CaM-binding domain (CaMBD, purple). (B) Predicted interaction of human VPS13D with CaM, colored as in (A). (C) Prediction of human VPS13B and CaM. The CaMBD-equivalent region between RBG 1 and RBG 2 , is highlighted in purple. CaM is not predicted to interact with any part of VPS13B shown here. (D) Predicted interaction of Sc VPS13 with Sc Cdc31. Sc Cdc31 (orange) differs from human CaM by an additional N-terminal helix (red). (E) Predicted interaction of Ct VPS13 with Ct Cdc31. Similarly, Ct Cdc31 (orange) differs from human CaM by an additional N-terminal loop (red). (F) When overexpressed in Expi293F cells, Ct VPS13 co-purifies with CaM in FLAG-IP. The binding of calmodulin to Ct VPS13 is unaffected by the presence of calcium or EGTA throughout the purification. Because of the difference in sizes for Ct VPS13 and CaM, the same samples were run twice on different gels (4-20% Tris-glycine and 3-8% Tris-acetate) to best resolve both proteins.

Article Snippet: Membranes were blocked for 1 hour at room temperature in 3% BSA prepared in Tris-buffered saline containing 0.1% Tween-20 (TBST), then incubated overnight at 4 °C with the anti-calmodulin antibody (Cat. #HY-P82082, MCE), 1:1000 diluted in 3% BSA and TBST.

Techniques: Binding Assay, Purification

Journal: bioRxiv

Article Title: Insights into the regulation of VPS13 family bridge-like lipid transfer proteins from the structure of VPS13C

doi: 10.1101/2025.11.10.687702

Figure Lengend Snippet: ( A ) Size-exclusion chromatography of VPS13C WT and calmodulin-binding defective mutants. Mutants elute with broader and shifted peaks compared to WT, yet remain soluble and do not aggregate in the void volume. ( B ) Like W395C, additional mutants M392A/W393D/W395K/I398D and W374K/W393D/W395K do not bind CaM, ( C ) lose the rod-like shape of WT protein but remain not non-aggregated, ( D ) and fail to localize to membrane contact sites upon LLOME treatment. ( E ) All CaM-binding defective mutants are expressed at levels comparable to VPS13C WT when overexpressed in Expi293F cells.

Article Snippet: Membranes were blocked for 1 hour at room temperature in 3% BSA prepared in Tris-buffered saline containing 0.1% Tween-20 (TBST), then incubated overnight at 4 °C with the anti-calmodulin antibody (Cat. #HY-P82082, MCE), 1:1000 diluted in 3% BSA and TBST.

Techniques: Size-exclusion Chromatography, Binding Assay, Membrane

Journal: Frontiers in Pharmacology

Article Title: Farnesyl Transferase Inhibitor Lonafarnib Enhances α7nAChR Expression Through Inhibiting DNA Methylation of CHRNA7 and Increases α7nAChR Membrane Trafficking

doi: 10.3389/fphar.2020.589780

Figure Lengend Snippet: Role of DNA methylation in the lonafarnib affected activity, total expression, membrane expression, and phosphorylation of a7nAChR. (A) Levels of a7nAChR total proteins in the hippocampus of control and lonafarnib-treated mice treated with vehicle, DNMT inhibitor RG108 or anisomycin. ** P < 0.01 vs. control mice, ## P < 0.01 vs. lonafarnib-treated mice (two-way ANOVA, followed by Tukey’s multiple comparison test). (B) Evoked I ACh by ACh (3 mM) in the slices of control and lonafarnib-treated mice treated with vehicle, RG108, or anisomycin, ** P < 0.01 vs. control mice; # P < 0.05 vs. lonafarnib-treated mice (two-way ANOVA, followed by Tukey’s multiple comparison test). (C) Levels of biotinylated a7nAChR (membrane surface) protein in the hippocampus of control and lonafarnib-treated mice treated with vehicle, RG108, or anisomycin. Surface a7nAChR was normalized by surface GluR2 protein, which was again normalized by vehicle-treated control group. ** P < 0.01 vs. control mice; # P < 0.05 vs. lonafarnib-treated mice (two-way ANOVA, followed by Tukey’s multiple comparison test). (D) Levels of phospho-a7nAChR in the hippocampus of control and lonafarnib-treated mice treated with vehicle, RG108, or anisomycin. The expression of protein and the amplitude of evoked I ACh were normalized by the values of control group with vehicle.

Article Snippet: CaMKII inhibitor KN93 and DNMT inhibitor RG108 were purchased from MCE.

Techniques: DNA Methylation Assay, Activity Assay, Expressing, Membrane, Phospho-proteomics, Control, Comparison

Journal: Frontiers in Pharmacology

Article Title: Farnesyl Transferase Inhibitor Lonafarnib Enhances α7nAChR Expression Through Inhibiting DNA Methylation of CHRNA7 and Increases α7nAChR Membrane Trafficking

doi: 10.3389/fphar.2020.589780

Figure Lengend Snippet: Lonafarnib administration affects CaMKII signaling pathways, partially modulating the membrane expression of a7nAChR. (A and B) Levels of phospho-PKC and phospho-PKA in the hippocampus of control and lonafarnib-treated mice treated with vehicle, RG108, or anisomycin. (C) Levels of phospho-CaMKII in the hippocampus of control and lonafarnib-treated mice treated with vehicle, RG108, or anisomycin. ** P < 0.01 vs. control mice (two-way ANOVA, followed by Tukey’s multiple comparison test). (D) Levels of biotinylated a7nAChR (membrane) protein in the hippocampus of control and lonafarnib-treated mice treated with vehicle or CaMKII pathway blocker KN93. Surface a7nAChR was normalized by surface GluR2 protein, which was again normalized by vehicle-treated control group. ** P < 0.01 vs. control mice; # P < 0.05 vs. lonafarnib-treated mice (two-way ANOVA, followed by Tukey’s multiple comparison test). (E) Levels of a7nAChR total proteins in the hippocampus of control and lonafarnib-treated mice treated with vehicle and KN93, and total a7nAChR was normalized by GAPDH, which was again normalized by vehicle-treated group. ** P < 0.01 vs. control mice (two-way ANOVA, followed by Tukey’s multiple comparison test). The expression of protein was normalized by the values of control group with vehicle.

Article Snippet: CaMKII inhibitor KN93 and DNMT inhibitor RG108 were purchased from MCE.

Techniques: Protein-Protein interactions, Membrane, Expressing, Control, Comparison

Journal: BMC Nephrology

Article Title: Intrarenal microRNA signature related to the fibrosis process in chronic kidney disease: identification and functional validation of key miRNAs

doi: 10.1186/s12882-019-1512-x

Figure Lengend Snippet: Overexpression of miRNA mimics inhibits luciferase reporter activity in HK-2 cells. a The schematic luciferase reporter constructs. A mutant construct was made by replacing four nucleotides in the miRNA seed binding site of target gene 3’UTR. Mutated nucleotides were marked in gray and underlined. The luciferase reporter assay of ITGB8 ( b ) and CALM3 ( c ) 3’UTR reporter in HK-2 cells at 48 h after transfection. EV: empty vector; WT: wild-type; Mut: mutant; NC: scrambled negative control transfection.* p < 0.05 versus NC. Data represent mean ± SEM for at least three independent experiments

Article Snippet: The primary antibodies used in this study included ITGB8 (SC-25714, Santa Cruz Biotechnology) and CALM3 (NBP2–15669, Novus Biologicals).

Techniques: Over Expression, Luciferase, Activity Assay, Construct, Mutagenesis, Binding Assay, Reporter Assay, Transfection, Plasmid Preparation, Negative Control

Journal: Technology in cancer research & treatment

Article Title: A Pyroptosis-Related Gene Signature Predicts Prognosis and Tumor Immune Microenvironment in Colorectal Cancer.

doi: 10.1177/15330338241277584

Figure Lengend Snippet: Figure 6. The PRGs protein expression as analyzed by IHC. The UBE2J2, NADK, GABRD, CALML6, PLCH2 and AGRN expression were assessed in CRC and para-carcinoma specimens by using Immuno-histochemistry, the Magnification is ×400, Scale bars indicate 50 μm.

Article Snippet: SAB4301032; 1:100 dilution; SIGMA), UBE2J2(cat.no.433663-1;1:100 dilution; Signalway antibody), CALML6 (cat.no.22274-1-AP; 1:100 dilution; Proteintech), PLCH2(cat.no.DF9032; 1:100 dilution; Affinity biosciences), primary antibodies were incubated with slides for overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: Dalpiciclib and Pyrotinib Exert Synergistic Antitumor Effects in Triple Positive Breast Cancer

doi: 10.1101/2021.10.04.463019

Figure Lengend Snippet: a-b: Total ER expression and nuclear ER expression in BT474 cells treated with different drugs. (This assay was performed in triplicates independently). c: The introduction of dalpiciclib to pyrotinib could significantly decrease the total and nuclear expression of ER, thus reversed the ER activation caused by pyrotinib and CALML5 could be served as a potential marker of ER activation after the treatment of pyrotinib.

Article Snippet: After these preparation procedures, the sections of each sample were incubated with the primary anti-ER antibody (Abcam Company, ab32063), anti-HER2 antibody (Abcam Company, ab134182), and anti-CALML5 antibody (Proteintech, 13059-1-AP) at 4 °C overnight.

Techniques: Expressing, Activation Assay, Marker

Journal: bioRxiv

Article Title: Dalpiciclib and Pyrotinib Exert Synergistic Antitumor Effects in Triple Positive Breast Cancer

doi: 10.1101/2021.10.04.463019

Figure Lengend Snippet: a: Western blot analysis of HER2 signaling pathway and cell cycle pathway in BT474 cells treated with different drugs or their combination. (This assay was performed in triplicates independently). b: Cell cycle analysis in BT474 cells treated with different drugs or their combination. (Data was presented as mean ±SDs, *** P <0.001 using Student’s t -test; all the assays were performed independently in triplicates). c: Representative views of CALML5 positive/negative tissue. The difference of PR+PCR ratio and PD+SD ratio in patients who received anti-HER2 therapy plus chemotherapy or anti-HER2 therapy plus CDK4/6 inhibitor regarding on their expression of CALML5. (**** P <0.0001 using Student’s t -test). d: Representative views of CALML5 positive/negative tissue. Ratio of patients with elevated or decreased CALML5 after receiving anti-HER2 therapy plus chemotherapy, or anti-HER2 therapy plus CDK4/6 inhibitor. (**** P <0.0001 using Student’s t -test).

Article Snippet: After these preparation procedures, the sections of each sample were incubated with the primary anti-ER antibody (Abcam Company, ab32063), anti-HER2 antibody (Abcam Company, ab134182), and anti-CALML5 antibody (Proteintech, 13059-1-AP) at 4 °C overnight.

Techniques: Western Blot, Cell Cycle Assay, Expressing