Journal: Research in Pharmaceutical Sciences

Article Title: Enhancing soluble expression and purification of a bispecific scFv antibody in Escherichia coli using chaperones

doi: 10.4103/RPS.RPS_10_24

Figure Lengend Snippet: (A) Schematic presentation of the pET28a-scFv-BsAb expression vector; (B) SDS-PAGE and western blot analysis of bacterial expression of histidine-tagged scFv-BsAb with or without chaperones. Lane 1: without chaperone before induction; lanes 2-4: without chaperone after induction by IPTG (0.25, 0.5, and 1 mM, respectively); lanes 5, 7, and 9: co-expression with chaperones before induction; lanes 6, 8, and 10: co-expression with chaperones after induction by IPTG (0.25, 0.5, and 1 mM, respectively); lane M: Thermofisher protein molecular weight marker 26610; Lane 11: western blot analysis using anti-His-HRP antibody. scFv, Single-chain variable fragment; IPTG, isopropyl-β-D-1-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Article Snippet: To confirm the protein expression and compare the soluble expression level in each condition, the samples were loaded on 12% sodium dodecyl sulfate-polyacrylamide gels, and electrophoresis was performed (SDS-PAGE) with a constant voltage of 150 V. Also, to confirm the expression of the His-tagged scFv-BsAb, the whole protein sample, which was first analyzed by SDS-PAGE, was transferred to polyvinylidene difluoride (PVDF) membranes, and western blotting was done using anti-His (C-term)-horseradish peroxidase (HRP) antibody (Invitrogen, USA).

Techniques: Expressing, Plasmid Preparation, SDS Page, Western Blot, Molecular Weight, Marker, Polyacrylamide Gel Electrophoresis

Journal: Research in Pharmaceutical Sciences

Article Title: Enhancing soluble expression and purification of a bispecific scFv antibody in Escherichia coli using chaperones

doi: 10.4103/RPS.RPS_10_24

Figure Lengend Snippet: Fluorescence intensity histogram based on the results of flow cytometry of PBMCs and HEK293 cells after incubation with scFv-BsAb and a secondary antibody (anti-His-FITC). The lymphocyte population in PBMCs was gated based on forward scatter and side scatter. scFv, Single-chain variable fragment.

Article Snippet: To confirm the protein expression and compare the soluble expression level in each condition, the samples were loaded on 12% sodium dodecyl sulfate-polyacrylamide gels, and electrophoresis was performed (SDS-PAGE) with a constant voltage of 150 V. Also, to confirm the expression of the His-tagged scFv-BsAb, the whole protein sample, which was first analyzed by SDS-PAGE, was transferred to polyvinylidene difluoride (PVDF) membranes, and western blotting was done using anti-His (C-term)-horseradish peroxidase (HRP) antibody (Invitrogen, USA).

Techniques: Fluorescence, Flow Cytometry, Incubation

Journal: bioRxiv

Article Title: Desmoglein-3 modulates p38MAPK and ERK signaling responses through the mechano-sensitive channel Piezo1

doi: 10.64898/2026.05.11.723746

Figure Lengend Snippet: A . Immunofluorescence staining for DSG3 and desmoplakin (DSP) of CTRL and DSG3-KO HaCaT keratinocytes. Scale bar 10 μm. B . Quantification of DSP intensity over the corresponding membrane length (μm), Mann-Whitney test. Each data point represents one cell from three independent experiments in total. C . Immunofluorescence staining for Pan-cytokeratin (pan-CK) of CTRL and DSG3-KO HaCaT keratinocytes. Scale bar 10 μm. D . Analysis of pan-CK staining by quantifying the intensity (a.u) perpendicular to cell borders over a distance of 10 µm. E . Dispase-based dissociation assay of CTRL and DSG3-KO cells. Representative images and quantifications of N = 3 are shown, Welch’s t-test. F-G . Western blot and representative quantification of phosphorylated p38MAPK (p-p38MAPK) and ERK (pERK) in CTRL and DSG3-KO HaCaT keratinocytes. Each dot represents biological replicates. Welch’s t-test.

Article Snippet: The following primary antibodies were diluted with Odyssey blocking buffer in TBS containing 0.1% Tween 20 (Thermo Fisher Scientific) and incubated overnight at 4°C, with rotation: mouse GAPDH mAb (Santa Cruz Biotechnology, #sc-47724), rabbit phospho-p38MAPK Thr180/Tyr182 (Cell Signaling Technology, #4511S), rabbit p38MAPK (Cell Signaling Technology, #9212S), rabbit ERK 1/2 (p44/42) (Cell Signaling Technology, #9102), phospho-ERK (Santa Cruz, sc-7383), mouse plakoglobin (Progen, #61005), mouse desmoplakin 1/2 (Progen, #61003), rabbit Piezo1 (Proteintech, # 15939-1-AP), rabbit Desmoglein-3 (5G11, Santa Cruz Biotechnology, # sc-53487), Desmoglein-2 (10G11, Acris OriGene, #BM5016), mouse Pan Cytokeratin mAb (Thermo Fisher, #41-9003-82), Phalloidin (Thermo Fisher, #21833).

Techniques: Immunofluorescence, Staining, Membrane, MANN-WHITNEY, Western Blot

Journal: bioRxiv

Article Title: Loss of dystrophin reduces CB1 receptor expression and endocannabinoid-dependent synaptic plasticity in the cerebellar cortex

doi: 10.64898/2026.03.20.713279

Figure Lengend Snippet: A. Representative Pinceaux staining showing a PC Soma (green, calbindin) along with the characteristic pinceaux formation on the axon side (red, CB1R) in both genotypes. B. Quantification of average CB1R pinceaux intensity across both genotypes and slide sets (VGAT, VGLUT1, VGLUT2). C. Same as B, but area of pinceaux. D-F. Proportion of CB1R puncta that colocalize with the respective vesicular marker before and after a 100 pixel shift of the CB1R channel along the X axis.

Article Snippet: Slides were incubated for 48 hours at 4°C under agitation with the following primary antibodies in blocking buffer: calbindin (1:3000, Sigma-Aldrich, Cat# C9848), CB1R (1:1000, Cayman Chemical Company, Cat# 10006590), and one of the following vesicular transporters - VGAT (1:250, Synaptic Systems, Cat# 131004), VGLUT2 (1:500, Synaptic Systems, Cat# 135418), or VGLUT1 (1:5000, Synaptic Systems, Cat# 135304).

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: Loss of dystrophin reduces CB1 receptor expression and endocannabinoid-dependent synaptic plasticity in the cerebellar cortex

doi: 10.64898/2026.03.20.713279

Figure Lengend Snippet: A. Representative confocal images from WT, DMD mdx and CB1R KO cerebella demonstrating calbindin (green), raw CB1R (red), and filtered CB1R puncta (see methods: image analysis). The right column shows an expanded and merged view of the area indicated in the filtered CB1R column. Quantification of mean CB1R puncta intensity ( B ), density ( C ), and area ( D ) in WT (black) and DMDmdx (blue) cerebella.

Article Snippet: Slides were incubated for 48 hours at 4°C under agitation with the following primary antibodies in blocking buffer: calbindin (1:3000, Sigma-Aldrich, Cat# C9848), CB1R (1:1000, Cayman Chemical Company, Cat# 10006590), and one of the following vesicular transporters - VGAT (1:250, Synaptic Systems, Cat# 131004), VGLUT2 (1:500, Synaptic Systems, Cat# 135418), or VGLUT1 (1:5000, Synaptic Systems, Cat# 135304).

Techniques:

Journal: bioRxiv

Article Title: Loss of dystrophin reduces CB1 receptor expression and endocannabinoid-dependent synaptic plasticity in the cerebellar cortex

doi: 10.64898/2026.03.20.713279

Figure Lengend Snippet: A. Representative images demonstrating calbindin (green), filtered CB1R (red), and filtered VGAT (cyan) staining across WT and DMD mdx cerebella. The right column shows an expanded and merged view of the area indicated in the VGAT image. Quantification of mean intensity ( B ), density ( C ), and area ( D ) of VGAT puncta in the molecular layer. E. Percentage of VGAT puncta colocalized with CB1R puncta. F. Intensity of CB1R puncta colocalized with VGAT puncta. G. Intensity of CB1R puncta not colocalized with VGAT puncta.

Article Snippet: Slides were incubated for 48 hours at 4°C under agitation with the following primary antibodies in blocking buffer: calbindin (1:3000, Sigma-Aldrich, Cat# C9848), CB1R (1:1000, Cayman Chemical Company, Cat# 10006590), and one of the following vesicular transporters - VGAT (1:250, Synaptic Systems, Cat# 131004), VGLUT2 (1:500, Synaptic Systems, Cat# 135418), or VGLUT1 (1:5000, Synaptic Systems, Cat# 135304).

Techniques: Staining

Journal: bioRxiv

Article Title: Loss of dystrophin reduces CB1 receptor expression and endocannabinoid-dependent synaptic plasticity in the cerebellar cortex

doi: 10.64898/2026.03.20.713279

Figure Lengend Snippet: A. Representative images demonstrating calbindin (green), filtered CB1R (red), and filtered VGLUT1 (cyan) staining across WT and DMD mdx cerebella. The right column shows an expanded and merged view of the area indicated in the VGLUT1 image. Quantification of mean intensity ( B ), density ( C ), and area ( D ) of VGLUT1 puncta in the molecular layer. E. Percentage of VGLUT1 puncta associated with CB1R puncta. F. Intensity of CB1R puncta colocalized with VGLUT1 puncta. G. Intensity of CB1R puncta not colocalized with VGLUT1 puncta

Article Snippet: Slides were incubated for 48 hours at 4°C under agitation with the following primary antibodies in blocking buffer: calbindin (1:3000, Sigma-Aldrich, Cat# C9848), CB1R (1:1000, Cayman Chemical Company, Cat# 10006590), and one of the following vesicular transporters - VGAT (1:250, Synaptic Systems, Cat# 131004), VGLUT2 (1:500, Synaptic Systems, Cat# 135418), or VGLUT1 (1:5000, Synaptic Systems, Cat# 135304).

Techniques: Staining

Journal: bioRxiv

Article Title: Loss of dystrophin reduces CB1 receptor expression and endocannabinoid-dependent synaptic plasticity in the cerebellar cortex

doi: 10.64898/2026.03.20.713279

Figure Lengend Snippet: A. Representative images demonstrating calbindin (green), filtered CB1R (red), and filtered VGLUT2 (cyan) staining across WT and DMD mdx cerebella. The right column shows an expanded and merged view of the area indicated in the VGLUT2 image. Quantification of mean intensity ( B ), density ( C ), and area ( D ) of VGLUT2 puncta in the molecular layer. E. Percentage of VGLUT2 puncta associated with CB1R puncta. F. Average intensity of CB1R puncta colocalized with VGLUT2 puncta. G. Intensity of CB1R puncta not colocalized with VGLUT2 puncta.

Article Snippet: Slides were incubated for 48 hours at 4°C under agitation with the following primary antibodies in blocking buffer: calbindin (1:3000, Sigma-Aldrich, Cat# C9848), CB1R (1:1000, Cayman Chemical Company, Cat# 10006590), and one of the following vesicular transporters - VGAT (1:250, Synaptic Systems, Cat# 131004), VGLUT2 (1:500, Synaptic Systems, Cat# 135418), or VGLUT1 (1:5000, Synaptic Systems, Cat# 135304).

Techniques: Staining

Journal: Molecular Medicine

Article Title: Progressive endocannabinoid system dysregulation in autosomal dominant polycystic kidney disease

doi: 10.1186/s10020-026-01457-w

Figure Lengend Snippet: ECS components are progressively dysregulated in human ADPKD kidney tissue. a Microarray analysis ( GSE7869 ) of human kidney tissue shows stepwise increases in CNR1 transcript from healthy cortex to minimally cystic (PKDm) and fully cystic (PKD) ADPKD tissue, with corresponding reductions in AEA-metabolizing enzymes NAPEPLD and FAAH . b Single-nucleus RNA-sequencing (snRNA-seq) analysis of human ADPKD kidneys ( n = 8) versus healthy controls ( n = 5) demonstrate consistent CNR1 upregulation and marked downregulation of NAPEPLD and FAAH , while 2-AG-metabolizing enzymes remain largely unchanged. c snRNA-seq analysis of diabetic kidney disease (DKD; n = 5 patients; controls n = 6) reveals minimal alterations in CNR1 and ECS-metabolizing enzymes. d Gene expression analysis by qPCR confirms CNR1 upregulation in human ADPKD kidney tissue ( n = 17) versus non-cystic nephrectomy controls ( n = 5), with concurrent changes in ECS enzyme transcription. e-i eCB quantification by liquid chromatography-tandem mass spectrometry (LC–MS/MS) reveals significant depletion of tissue anandamide (AEA); e , N -oleoylethanolamine (OEA); f , 2-arachidonoylglycerol (2-AG); h , and arachidonic acid (AA); i , while N -palmitoylethanolamine (PEA); g remains unchanged. j-l Western blot analysis shows substantial inter-individual variability in CB 1 R protein levels without significant difference between ADPKD ( n = 6) and control kidneys ( n = 4), but significant reductions in DAGLα/β, NAPEPLD, MGLL and FAAH protein. Western blots were normalized to total proteins. Data represent mean ± SEM. Statistics of control versus PKD represented by * and control versus PKDm by #. Statistical significance assessed by Mann–Whitney U test or unpaired t -test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Sections were stained with rabbit anti-CB 1 R antibody (1:200, 10006590, Cayman) followed by a goat anti-rabbit HRP conjugate (ImmPRESSTM, Vector laboratories).

Techniques: Microarray, RNA Sequencing, Gene Expression, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, MANN-WHITNEY

Journal: Molecular Medicine

Article Title: Progressive endocannabinoid system dysregulation in autosomal dominant polycystic kidney disease

doi: 10.1186/s10020-026-01457-w

Figure Lengend Snippet: Temporal endocannabinoid system dysregulation during Pkd1 RC/RC disease progression. a-g Quantitative PCR analysis of ECS-related genes across disease stages (9 and 12 months) showing progressive upregulation of Cnr1 a and Cnr2 b transcripts, with corresponding changes in ECS metabolic enzymes: Dagla c , Daglb d , Napepld e , Mgll f , and Faah g . h-n Western blot analysis and quantification of ECS proteins across disease stages, demonstrating sustained CB 1 R protein elevation at 9 and 12 months h, i , stable 2-AG-related enzyme proteins (DAGLα, DAGLβ, MGLL; h, j, k, m ), and stage-specific changes in AEA-related enzymes including elevated NAPEPLD at 12 months ( h, l ) and reduced FAAH at 9 months ( h, n ). Data represent mean ± SEM from WT ( n = 5) and Pkd1 RC/RC ( n = 6–7) per group per timepoint. Statistical analysis: unpaired t -test comparing Pkd1 RC/RC to age-matched WT. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus WT

Article Snippet: Sections were stained with rabbit anti-CB 1 R antibody (1:200, 10006590, Cayman) followed by a goat anti-rabbit HRP conjugate (ImmPRESSTM, Vector laboratories).

Techniques: Biomarker Discovery, Real-time Polymerase Chain Reaction, Western Blot