ccl2 recombinant protein  (R&D Systems)

Journal: Cell Death & Disease

Article Title: REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy

doi: 10.1038/s41419-025-07396-4

Figure Lengend Snippet: Diabetes was induced in REDD1 +/+ and REDD1 −/− mice by streptozotocin (STZ) administration. Non-diabetic control mice received vehicle (Veh). A REDD1 protein was evaluated in kidney cortical tissue homogenates by western blotting. Representative blots are shown. Molecular mass in kDa is indicated at right of each blot. B Correlation between fasting blood glucose and urine ACR is shown for REDD1 +/+ mice ( blue ; Pearson r = 0.72; p < 0.0001) and REDD1 −/− mice ( red ; Pearson r = 0.55; p = 0.029). C Ccl2 mRNA expression was quantified in kidney homogenates by qPCR. D CCL2 protein abundance was quantified in kidney homogenates by western blotting. E Il1b mRNA expression was quantified in kidney homogenates by qPCR. F IL-1β protein levels were determined in kidney homogenates by western blotting and quantified by ELISA. Individual data points are plotted with values presented as means ± SD ( n = 4–6). Differences between groups were identified by two-way ANOVA. * p < 0.05 versus Veh; # p < 0.05 versus REDD1 +/+ . n.d., not detected.

Article Snippet: CCL2 recombinant protein (R&D systems, Minneapolis, MN, USA) was subjected to western blotting and CCL2 protein in cell (10 5 cells) and tissue lysates were quantified (Fig. S ).

Techniques: Control, Western Blot, Expressing, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy

doi: 10.1038/s41419-025-07396-4

Figure Lengend Snippet: A , B Diabetes was induced in REDD1 +/+ and REDD1 −/− mice by administration of streptozotocin (STZ). Non-diabetic control mice received vehicle (Veh). A Nuclear isolates were prepared from kidney homogenates. NF-κB and Lamin B were examined in nuclear isolates by western blotting and NF-κB activity was quantified by DNA-binding ELISA. Representative blots are shown with protein molecular mass in kDa indicated at right of each blot. B REDD1 ( red ) and Nephrin ( green ) were visualized in kidneys by immunofluorescence microscopy. White box indicates area shown at increased magnification. Representative micrographs are shown (scale bar 50 μm). C – I Wild-type (WT) and REDD1 knockout (KO) CIHP-1 were exposed to culture media containing either 30 mM glucose (HG) or 5 mM glucose plus 25 mM mannitol (OC) for 48 h. NF-κB phosphorylation at S536 and REDD1 protein abundance was determined in cell lysates by western blotting ( C ). Nuclear localization of NF-κB p65 ( white arrowheads ) was evaluated by immunofluorescence ( D ). Nuclei were visualized with DAPI (scale bar 25 μm). NF-κB activity was measured in lysates from cells expressing NF-κB firefly luciferase/ Renilla luciferase reporter plasmids by dual luciferase assay ( E ). Relative expression of IL1B and CCL2 mRNA were determined by qPCR ( F ). IL-1β secreted into culture media was determined by ELISA ( G ). Chromatin immunoprecipitation (ChIP)-PCR analysis was carried out in WT and REDD1 KO podocytes to determine binding of p65 NF-κB to the promoter region of the CCL2 gene ( H ). CCL2 protein levels were determined in cell lysates by western blotting ( I ). J NF-κB p65 phosphorylation and NF-κB luciferase reporter activity was evaluated in REDD1 KO cells expressing either an empty vector control (EV) or hemagglutinin (HA)-tagged REDD1. Individual data points are presented as means ± SD ( n = 4–6). Differences between groups were identified by two-way ANOVA. * p < 0.05 versus Veh or NG; # p < 0.05 versus REDD1 +/+ , WT, or EV.

Article Snippet: CCL2 recombinant protein (R&D systems, Minneapolis, MN, USA) was subjected to western blotting and CCL2 protein in cell (10 5 cells) and tissue lysates were quantified (Fig. S ).

Techniques: Control, Western Blot, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Microscopy, Knock-Out, Phospho-proteomics, Quantitative Proteomics, Expressing, Luciferase, Chromatin Immunoprecipitation, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy

doi: 10.1038/s41419-025-07396-4

Figure Lengend Snippet: A , B Differentiated wild-type (WT) and REDD1 knockout (KO) CIHP-1 were exposed to culture media containing either 30 mM glucose (HG) or 5 mM glucose plus 25 mM mannitol as an osmotic control (OC) for 48 h. Transwell migration assay was used to evaluate chemotaxis in a co-culture model with CIHP-1 and THP-1 macrophages ( A ). Macrophages were stained with crystal violet and cells that migrated across the Transwell were counted ( B ). C Cre-lox recombination was used to achieve conditional podocyte-specific REDD1 knockout (REDD1 PodKO). D – H Diabetes was induced in REDD1 fl/fl and REDD1 PodKO mice by streptozotocin (STZ) administration. Non-diabetic groups were administered a vehicle (Veh) control. All assessments were performed after 16 weeks of diabetes. Urine albumin to creatinine ratio (ACR) was determined (D). Kidney sections from diabetic and non-diabetic mice were immunolabeled for REDD1 ( red ) and the podocyte marker Nephrin ( green ) ( E ). Protein abundance of CCL2 was determined in renal homogenates by western blotting ( F ). Representative blots are shown with protein molecular mass in kDa indicated at right of each blot. Kidney sections were immunolabelled for F4/80 ( red ) and nuclei were counterstained with Hoechst 33342 ( blue ) ( G ). Representative micrographs (scale bar 50 µm) are shown. Immune cell populations of CD11b + F4/80+ macrophages ( H ) and CD86 + M1 macrophages ( I ) were determined by flow cytometry. Individual data points are plotted. Significance was analyzed by two-way ANOVA and pairwise comparisons were made using the Tukey’s test for multiple comparisons. * p < 0.05 versus OC or Veh; #, p < 0.05 versus WT or REDD1 fl/fl .

Article Snippet: CCL2 recombinant protein (R&D systems, Minneapolis, MN, USA) was subjected to western blotting and CCL2 protein in cell (10 5 cells) and tissue lysates were quantified (Fig. S ).

Techniques: Knock-Out, Control, Transwell Migration Assay, Chemotaxis Assay, Co-Culture Assay, Staining, Immunolabeling, Marker, Quantitative Proteomics, Western Blot, Flow Cytometry

Journal: Pharmacological research

Article Title: Discovery of a CCR2-targeting pepducin therapy for chronic pain.

doi: 10.1016/j.phrs.2024.107242

Figure Lengend Snippet: Fig. 1. CCR2-targeting pepducins selectively inhibit CCL2-induced Gαi1/GαoA activation and β-arrestin2 recruitment through allosteric modulation. (A) Rational design of the pepducins derived from ICL1 (PP101) and ICL3 (PP103, PP104) of CCR2 and their respective amino acid sequences (B-C). Time course of Gαi1 and GαoA activation upon CCL2 (2 µM) or pepducin (10 µM) stimulation assessed by measuring the BRET2 signal in real-time in HEK293 cells transiently transfected with rCCR2 and BRET2-based G-protein biosensors (D-E) (n = 5 duplicate). Antagonist effects of PP101, PP103 or PP104 on CCL2-induced Gαi1 or GαoA activation and β-arrestin 2 recruitment (F-H) (n = 3 duplicate). BRET2 ratios were normalized according to CCL2; values for HEK293 cells treated with CCL2 were set to 100 % activation and the non-stimulated cells were set at 0 %. Cross-antagonism of PP101 in Gi/o-protein inhibition at CXCR4, MOR, NTS1 or CCR5 stimulated respectively by SDF-1, DAMGO, NT(8−13) and rCCL5 were also assessed (I-L) (n = 3, quadruplicate). Palm: palmitate. Data are represented as mean ± SEM.

Article Snippet: In the first protocol (a time-course experiment), the cells were stimulated with 100 nM CCL2 (R&D Systems # 3144-JE-025/ CF) or 10 mM pepducin, then stimulated with the coelenterazine 400 A substrate (5 mM) (Gold Biotechnology #C-320–1) and read for 30 min on Mithras2 Plate Reader (Berthold) using a BRET2 filter set (400–450 nm and 500–550 nm emission filters).

Techniques: Activation Assay, Derivative Assay, Transfection, Inhibition

Journal: Pharmacological research

Article Title: Discovery of a CCR2-targeting pepducin therapy for chronic pain.

doi: 10.1016/j.phrs.2024.107242

Figure Lengend Snippet: Fig. 2. The pepducin PP101 prevents the nociceptive behaviors induced by bone cancer without deleterious effect on bone remodeling and tumor growth. (A) Effect of CCL2 (1 µg/rat, i.t.) and co-injection of CCL2 with PP101 (125 nmol/rat, i.t.) on mechanical thresholds, compared to rats receiving SCR101 control pepducins. (B) Paw withdrawal thresholds measured at day 3 following i.t. saline (n = 14), CCL2 (n = 6), CCL2+SCR101 (n = 7), CCL2+PP101 (n = 8) and CCL2+RS 504393, a selective CCR2 antagonist (60 nmol/rat; n = 9). (C) Schematic representation of the study design and of the behavioral tests used to measure the stimulus- evoked and non-evoked pain-related behaviors in tumor-bearing female rats. Repeated daily administrations of PP101 or SCR101 (125 nmol/rat, i.t.) between days 11 and 14 on (D) paw retroflexion score, (E) burrowing behavior, (F and G) dynamic weight-bearing and (H-I) mechanical allodynia, (sham+SCR101 n =7, can cer+SCR101 n = 7, cancer+PP101 n = 6). (J-O) Tomodensitometry (µCT) analysis of the bone structure of sham-operated and tumor-implanted femur (sham+SCR101 n = 7, cancer+SCR101 n = 8, cancer+PP101 n = 6). (P) Volume of the tumor on the ipsilateral femur (sham+SCR101 n = 4, cancer+SCR101 n = 5, cancer+PP101 n = 8). Data are represented as mean ± SEM. Two-way ANOVA followed by Sidak’s multiple comparisons test in (A, D-H), Kruskal-Wallis followed by Dunn’s multiple comparisons test in (B, L-P). *P < 0.05, **P < 0.01, ***P < 0.001. # compared with sham+SCR101 and * compared with cancer+SCR101.

Article Snippet: In the first protocol (a time-course experiment), the cells were stimulated with 100 nM CCL2 (R&D Systems # 3144-JE-025/ CF) or 10 mM pepducin, then stimulated with the coelenterazine 400 A substrate (5 mM) (Gold Biotechnology #C-320–1) and read for 30 min on Mithras2 Plate Reader (Berthold) using a BRET2 filter set (400–450 nm and 500–550 nm emission filters).

Techniques: Injection, Control, Saline