rabbit anti clic5  (Alomone Labs)

Journal: Frontiers in Immunology

Article Title: Differential effect of lactate on synovial fibroblast and macrophage effector functions

doi: 10.3389/fimmu.2023.1183825

Figure Lengend Snippet: Lactate transporter expression in synovial compartment. (A) Representative IF staining of SLC16A1 (blue) and SLC16A3 (green) expression by synovial fibroblasts (CD90, red) and macrophages (CD68, red) from n=8 RA synovial tissues. (B) Quantification of the colocalization (Pearson’s correlation coefficient, ref 21) between SLC16A1 (blue) and SLC16A3 (green) and CD90 (red) and CD68 (red) within n=8 RA synovial tissues. Data are expressed as mean ± SEM. Anova test **p < 0.05; (C) SLC16A1 and SLC16A3 expression (red) by sub lining (CD90, green) and lining (CLIC5 or PRG4, blue) fibroblasts. (D) SLC16A1 and SLC16A3 expression (blue and green respectively) by sub lining (CD68 + TREM2 - , orange) and lining (CD68 + TREM2 + ) macrophages. Scale bar 50 um. (E) scRNAseq expression of lactate transporters in synovial cellular subsets: Fibroblast: sub-lining SC-F1 (CD34 + CD90 + ), SC-F2 (HLA-DRA high CD90 + ), SC-F3 (DKK + CD90 + ) and lining SC-F4 (CD55 + CD90 - PRG4 + CLIC5 + ) subsets (ref 1, 2). Macrophages: sub-lining MerTK neg TREM2 neg SC-M1 (IL-1b + CD14 + ), SC-M4 (IFN-activated), and lining MerTK pos TREM2 pos SC-M2 (NUPR1 + CD14 + ), SC-M3 (C1QA + CD14 + ) subsets (ref 2, 5). T cells: SC-T1 (CCR7 + ), SC-T2 (Treg cells), SC-T3 (Follicular helper T cells), SC-T4 (Granzyme K + ), SC-T5 (Granulysin + , Granzyme B + ), SC-T6 (Granzyme K + , Granzyme B + ). B cells: SC-B1 (Naïve), SC-B2 (Memory), SC-B3 (Autoimmune), SC-B4 (Plasmablasts) (ref 2). Synovial tissues were taken by RA (n=36) and OA (n=15) patients (AMP dataset,ref 1, 2).

Article Snippet: After antigen retrieval step (45 minutes) and block of non-specific binding (1 hour), paraffin-embedded tissue sections were incubated at 4°C (overnight or for 1hour) with rabbit polyclonal anti-SLC16A1 Ab (1:300, Bethyl), mouse monoclonal anti-SLC16A3 (1:100, Santa Cruz), polyclonal sheep anti-CD90 (1:100, R&D), biotin anti-CD68 (1:100, Novus), goat anti-TREM2 (1:100, Abcam), mouse anti-PRG4 (1:100, Novus), and rabbit anti-CLIC5 (1:100, ThermoFisher).

Techniques: Expressing, Staining

Journal: Experimental eye research

Article Title: The klotho-related protein KLPH (lctl) has preferred expression in lens and is essential for expression of clic5 and normal lens suture formation

doi: 10.1016/j.exer.2018.02.001

Figure Lengend Snippet: A) Western blot with anti-Clic5 for soluble and insoluble fractions of WT (+/+) and KO (−/−) lens extracts. Clic5 is present only in WT lens, mainly in the insoluble fraction of the lens extracts.

Article Snippet: Rabbit polyclonal anti-Clic5 antibody was obtained from Alomone Labs (Jerusalem, Israel); mouse anti-pericentrin antibody was obtained from Abcam (Cambridge, MA); goat polyclonal anti-RabIF antibody was purchased from Sigma Aldrich.

Techniques: Western Blot

Journal: Kidney international

Article Title: Proteomic analysis of the slit diaphragm complex: CLIC5 is a protein critical for podocyte morphology and function

doi: 10.1038/ki.2010.212

Figure Lengend Snippet: List of proteins identified in the slit diaphragm-enriched fraction

Article Snippet: The primary antibodies used were as follows: goat polyclonal anti-nephrin and goat polyclonal anti-podocalyxin (R&D Systems, Minneapolis, MN, USA); goat polyclonal anti-CLIC5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-CLIC5 and anti-transferrin receptor (Sigma, Saint Louis, MO, USA); anti-COX IV, anti-PSMA2, rabbit polyclonal anti-ERM, rabbit polyclonal anti-pERM and anti-flotillin 1 (Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal anti-podocin (Sigma); and goat anti-actin and rabbit anti-WT1 (Santa Cruz).

Techniques:

Journal: Kidney international

Article Title: Proteomic analysis of the slit diaphragm complex: CLIC5 is a protein critical for podocyte morphology and function

doi: 10.1038/ki.2010.212

Figure Lengend Snippet: Protein fractions produced during the biochemical isolation of SDs were immunoblotted with antibodies to nephrin and CLIC5. Nephrin and both isoforms of CLIC5 were identified in the final SD-enriched fraction, as well as in other fractions (antibodies are labeled to the right of each panel).

Article Snippet: The primary antibodies used were as follows: goat polyclonal anti-nephrin and goat polyclonal anti-podocalyxin (R&D Systems, Minneapolis, MN, USA); goat polyclonal anti-CLIC5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-CLIC5 and anti-transferrin receptor (Sigma, Saint Louis, MO, USA); anti-COX IV, anti-PSMA2, rabbit polyclonal anti-ERM, rabbit polyclonal anti-pERM and anti-flotillin 1 (Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal anti-podocin (Sigma); and goat anti-actin and rabbit anti-WT1 (Santa Cruz).

Techniques: Produced, Isolation, Labeling

Journal: Kidney international

Article Title: Proteomic analysis of the slit diaphragm complex: CLIC5 is a protein critical for podocyte morphology and function

doi: 10.1038/ki.2010.212

Figure Lengend Snippet: (a) Adult mouse kidney sections were double fluorescently labeled with (A) CLIC5 (red), (B) WT1 (green), a marker for podocytes, and (C) 4′,6-diamidino-2-phenylindole (DAPI) (blue), a nuclear marker. (D) A merged image that shows WT1 + podocytes colabeled with CLIC5 (arrowheads). CLIC5 was noted to localize to the cell body and cell processes (arrows) of podocytes. This is a representative fluorescence confocal image of a mouse glomerulus. (b) Immunoelectron microscopic localization of CLIC5 in adult mouse kidney. CLIC5 labeling was seen in foot processes with predominant localization in the apical and basal membranes and in regions proximal to slit diaphragms (arrowheads). CLIC5 also localized to glomerular endothelial cells (arrows). These are representative images taken at × 64,000–180,000 magnification.

Article Snippet: The primary antibodies used were as follows: goat polyclonal anti-nephrin and goat polyclonal anti-podocalyxin (R&D Systems, Minneapolis, MN, USA); goat polyclonal anti-CLIC5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-CLIC5 and anti-transferrin receptor (Sigma, Saint Louis, MO, USA); anti-COX IV, anti-PSMA2, rabbit polyclonal anti-ERM, rabbit polyclonal anti-pERM and anti-flotillin 1 (Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal anti-podocin (Sigma); and goat anti-actin and rabbit anti-WT1 (Santa Cruz).

Techniques: Labeling, Marker, Fluorescence

Journal: Kidney international

Article Title: Proteomic analysis of the slit diaphragm complex: CLIC5 is a protein critical for podocyte morphology and function

doi: 10.1038/ki.2010.212

Figure Lengend Snippet: (a) Primary podocyte cultures were labeled with (A) CLIC5 (red) and (B) actin (phalloidin, blue). (C) A merged image and (D) a magnified inset from the merged image showing that CLIC5 localized at the tips of actin filaments. This is a representative fluorescence confocal image of primary mouse podocytes after 14 days in vitro. (b) Three adjacent primary podocytes were fluorescently labeled and imaged with (A) CLIC5 (red), (B) merged with phalloidin (blue), (C) imaged with differential interference contrast, and (D) merged with differential interference contrast. CLIC5 had a punctate labeling pattern enriched at regions of cell–cell contact. Images were taken at × 60 magnification for (a) and at × 40 magnification for (b).

Article Snippet: The primary antibodies used were as follows: goat polyclonal anti-nephrin and goat polyclonal anti-podocalyxin (R&D Systems, Minneapolis, MN, USA); goat polyclonal anti-CLIC5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-CLIC5 and anti-transferrin receptor (Sigma, Saint Louis, MO, USA); anti-COX IV, anti-PSMA2, rabbit polyclonal anti-ERM, rabbit polyclonal anti-pERM and anti-flotillin 1 (Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal anti-podocin (Sigma); and goat anti-actin and rabbit anti-WT1 (Santa Cruz).

Techniques: Labeling, Fluorescence, In Vitro

Journal: Kidney international

Article Title: Proteomic analysis of the slit diaphragm complex: CLIC5 is a protein critical for podocyte morphology and function

doi: 10.1038/ki.2010.212

Figure Lengend Snippet: (a) Whole cellular lysates (WCLs) were produced from isolated glomeruli, and CLIC5A and CLIC5B expression levels were determined by western blot analysis. Actin immunoblotting served as a loading control to enable protein expression comparisons between samples. Expression of CLIC5A and CLIC5B decreased between CLIC5+/+, CLIC5+/−, and CLIC5−/− mice. There was no detectable expression of either CLIC5 isoform in CLIC5−/− mice. (b) Mouse kidney sections were double immunofluorescently labeled with CLIC5 (A and B) and WT1 (C and D) antibodies, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, merge, E and F). CLIC5+/+ glomeruli exhibited CLIC5 expression, whereas no CLIC5 was evident in kidney sections from CLIC5−/− mice. Immunofluorescence for CLIC5, WT1, and DAPI was merged, indicating that CLIC5 expression was absent in podocytes of CLIC5−/− mice. Images were taken at × 60 magnification.

Article Snippet: The primary antibodies used were as follows: goat polyclonal anti-nephrin and goat polyclonal anti-podocalyxin (R&D Systems, Minneapolis, MN, USA); goat polyclonal anti-CLIC5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-CLIC5 and anti-transferrin receptor (Sigma, Saint Louis, MO, USA); anti-COX IV, anti-PSMA2, rabbit polyclonal anti-ERM, rabbit polyclonal anti-pERM and anti-flotillin 1 (Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal anti-podocin (Sigma); and goat anti-actin and rabbit anti-WT1 (Santa Cruz).

Techniques: Produced, Isolation, Expressing, Western Blot, Labeling, Staining, Immunofluorescence

Journal: Kidney international

Article Title: Proteomic analysis of the slit diaphragm complex: CLIC5 is a protein critical for podocyte morphology and function

doi: 10.1038/ki.2010.212

Figure Lengend Snippet: (a) Western blot analysis was performed on whole cellular lysates (WCLs) derived from CLIC5+/+, CLIC5+/−, and CLIC5−/− glomeruli. pERM, total ERM, podocalyxin, and nephrin (labeled to the right of each blot) were detected using the appropriate antibodies, and immunoblotting for actin was performed to allow protein expression comparisons between each genotype. (b) To determine the relative protein expression levels, the signal intensities for ERM, podocalyxin, and nephrin were normalized to actin by densitometric analysis. To investigate whether the phosphorylation of the ERM protein complex differed between genotypes, a ratio of phosphorylated ERM to total ERM expression was calculated. Data were graphed as the mean ± range. No difference between genotypes was detected for the level of pERM compared with total ERM. However, there was more than a 1.5- and 2.5-fold decrease in the expression of total ERM in CLIC5+/− and CLIC5−/− mice, respectively, when compared with total ERM expression in CLIC5+/+ mice. Podocalyxin expression diminished by almost 1.5- and 3-fold for CLIC5+/− and CLIC5−/− mice, respectively. There was also a decrease in nephrin expression when CLIC5+/+ mice were compared with CLIC5−/− mice. (c) Images of glomeruli were obtained from kidney sections that were immunofluorescently labeled for CLIC5, pERM, podocalyxin, and nephrin. CLIC5+/+ glomeruli (A, C, E and G) and CLIC5−/− glomeruli (B, D, F and H).

Article Snippet: The primary antibodies used were as follows: goat polyclonal anti-nephrin and goat polyclonal anti-podocalyxin (R&D Systems, Minneapolis, MN, USA); goat polyclonal anti-CLIC5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-CLIC5 and anti-transferrin receptor (Sigma, Saint Louis, MO, USA); anti-COX IV, anti-PSMA2, rabbit polyclonal anti-ERM, rabbit polyclonal anti-pERM and anti-flotillin 1 (Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal anti-podocin (Sigma); and goat anti-actin and rabbit anti-WT1 (Santa Cruz).

Techniques: Western Blot, Derivative Assay, Labeling, Expressing

Journal: Kidney international

Article Title: Proteomic analysis of the slit diaphragm complex: CLIC5 is a protein critical for podocyte morphology and function

doi: 10.1038/ki.2010.212

Figure Lengend Snippet: (a) Glomeruli were isolated from CLIC5+/+ and CLIC5−/− kidneys, detergent extracts were produced from them, and CLIC5 was immunoprecipitated. Immunoblotting of podocalyxin (top panel), pERM (second panel), and total ERM (third panel) showed coimmunoprecipitation of CLIC5 with these proteins. Blots were reprobed with anti-CLIC5 to confirm immunoprecipitation of CLIC5 (fourth and fifth panels). Immunoblotting of supernatants with actin controlled for equal amounts of lysates analyzed, and immunoblotting the supernatants for podocalyxin, pERM, and ERM confirmed their presence in extracts from CLIC5+/+ and CLIC5−/− glomeruli. (b) Images of mouse whole glomeruli fixed and labeled with CLIC5 (A and D, red), pERM (B, green), podocalyxin (E, green), and merged images (C and F). (C) The pERM-CLIC5 merged image shows colocalization of these proteins that appeared as yellow labeling in the podocyte processes (magnified inset). Colocalization of CLIC5 with podocalyxin was also noted as yellow labeling in the merged image (F). Fluorescence confocal images were taken at × 60 magnification. (c) Primary podocyte cultures from CLIC5+/+ (A–C and G–I) and CLIC5−/− (D–F and J–L) mice were grown for 14 days in vitro before they were fluorescently labeled with CLIC5 (A, D, G, and J, red), pERM (B and E, green), podocin (H and K, green), and phalloidin-labeled actin filaments (blue); merged images (C, F, I, and L). CLIC5 labeling was only detected in CLIC5+/+ podocytes and was absent from CLIC5−/− podocytes. The merged image (C) indicated that CLIC5 colocalized with pERM and was most evident near edges and cell-cell contact regions of the podocytes. The merged image of CLIC5 with podocin (I) showed no colocalization.

Article Snippet: The primary antibodies used were as follows: goat polyclonal anti-nephrin and goat polyclonal anti-podocalyxin (R&D Systems, Minneapolis, MN, USA); goat polyclonal anti-CLIC5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-CLIC5 and anti-transferrin receptor (Sigma, Saint Louis, MO, USA); anti-COX IV, anti-PSMA2, rabbit polyclonal anti-ERM, rabbit polyclonal anti-pERM and anti-flotillin 1 (Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal anti-podocin (Sigma); and goat anti-actin and rabbit anti-WT1 (Santa Cruz).

Techniques: Isolation, Produced, Immunoprecipitation, Western Blot, Labeling, Fluorescence, In Vitro

Journal: Kidney international

Article Title: Proteomic analysis of the slit diaphragm complex: CLIC5 is a protein critical for podocyte morphology and function

doi: 10.1038/ki.2010.212

Figure Lengend Snippet: (a) Representative s.e.m. images of whole glomeruli from CLIC5+/+ mice (A) versus CLIC5−/− mice (B) at 4 weeks of age. These images were taken at × 2000 magnification. (C and D) Representative images of podocyte cell bodies and processes were taken at higher magnification, × 10,000 (C, CLIC5+/+ and D, CLIC5−/−). White arrowheads show the ‘longitudinal’ length of foot processes that were measured using ImageJ in b below. Images of transmission electron microscopy were taken at × 19,000 magnification. (b) Longitudinal lengths of foot processes in micrometers. Data are represented as mean ± s.e.m. from three to four mice of each genotype. A total of 140–180 processes were measured from each mouse (see methods for details.) *P-value <0.001 as compared with CLIC5+/+ mice. (c) Urine protein/creatinine ratios (mg/mg) were determined from male, 3-month-old CLIC5+/+, CLIC5+/−, and CLIC5−/− mice. Data were represented as mean ± s.e.m., n=4 per data group. **P-value <0.001 as compared with the CLIC5+/+ group.

Article Snippet: The primary antibodies used were as follows: goat polyclonal anti-nephrin and goat polyclonal anti-podocalyxin (R&D Systems, Minneapolis, MN, USA); goat polyclonal anti-CLIC5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-CLIC5 and anti-transferrin receptor (Sigma, Saint Louis, MO, USA); anti-COX IV, anti-PSMA2, rabbit polyclonal anti-ERM, rabbit polyclonal anti-pERM and anti-flotillin 1 (Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal anti-podocin (Sigma); and goat anti-actin and rabbit anti-WT1 (Santa Cruz).

Techniques: Transmission Assay, Electron Microscopy