Journal: Cell Communication and Signaling : CCS

Article Title: CLIP2: a novel functional player in AQP5 trafficking dynamics and implications for Sjögren’s disease

doi: 10.1186/s12964-025-02476-6

Figure Lengend Snippet: Identification of CLIP2 as a protein partner of AQP5. A , B Tandem mass spectra of CLIP2 peptide 634–645, ATLNSGPGAQQK, (+ 2 charged ion, m/z 586.4) from AQP5 IP samples in mouse parotid ( A ) and submandibular SG ( B ). Sequence specific b- and y-ions are labeled. C AQP5 immunoprecipitation (IP) performed using proteins from mouse SGs followed by WB detection using anti-CLIP2 antibodies. Input proteins from mouse submandibular glands (mSMG; lane A) and mouse parotid gland (mPG, lane B); IP performed using mSMG (lanes C, E) and mPG (lanes D, F) proteins in the presence (lanes C, D) or absence (negative controls; lanes E, F) of anti-AQP5 antibody; IP performed in the absence of any input proteins (additional negative control, lane G). D AQP5 IP performed using proteins from NS-SV-AC cells expressing AQP5 followed by WB detection using anti-CLIP2 antibodies. Input proteins from NS-SV-AC cells transfected with HA-hAQP5 (lane A) or SNAP-hAQP5 (lane B); IP performed using NS-SV-AC HA-AQP5 (lanes C, D) and NS-SV-AC SNAP-hAQP5 (lanes E, F) in the presence (lanes C, E) or in the absence (negative controls; lanes D, F) of anti-AQP5 antibody; IP performed in the absence of input proteins (additional negative control, lane G). Considering known batch-to-batch variation of commercial Sepharose-Protein A beads, whereby Protein A can shed from the beads under elution, the non-specific bands of ± 80–100 kDa ( C ) and ± 45–50 kDa ( D ) are likely non-specific bands corresponding in all likelihood to the IgG heavy chain (± 45–50 kDa) coupled or not to protein A (± 45 kDa) shed from the Sepharose beads.HA: hemagglutinin; SNAP: small protein derived from mammalian O6-alkylguanine-DNA-alkyltransferase

Article Snippet: The PVDF membranes were blocked for 1 h at RT with PBS containing 0.1% Tween 20 (PBS-T) and 5% skimmed milk, then incubated overnight at 4 °C with rabbit anti-CLIP2 antibody (1/1000, #25088-1-AP, Proteintech, Rosemont, IL, USA), rabbit anti-CLIP2 (1/1000, HPA020430, Sigma-Aldrich, St Louis, MI, USA), rabbit anti-AQP5 antibodies (1/1000, AB15858, Merck-Millipore, Burlington, MA, USA), mouse anti-β-actin (1/2000, #MAB1501, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Sequencing, Labeling, Immunoprecipitation, Negative Control, Expressing, Transfection, Derivative Assay

Journal: Cell Communication and Signaling : CCS

Article Title: CLIP2: a novel functional player in AQP5 trafficking dynamics and implications for Sjögren’s disease

doi: 10.1186/s12964-025-02476-6

Figure Lengend Snippet: Interaction between AQP5 and CLIP2 at the molecular level. A Binding curve from the MST-experiment showing the direct interaction between AQP5 and CLIP2. Data are expressed as the mean ± S.D. of bound fraction ( n = 3). The curve-line represents the curve fitting to a one-to-one binding model. B MST-data for the individual CLIP2-MTB domains (MTB1 and MTB2) showing the absence of interaction with AQP5

Article Snippet: The PVDF membranes were blocked for 1 h at RT with PBS containing 0.1% Tween 20 (PBS-T) and 5% skimmed milk, then incubated overnight at 4 °C with rabbit anti-CLIP2 antibody (1/1000, #25088-1-AP, Proteintech, Rosemont, IL, USA), rabbit anti-CLIP2 (1/1000, HPA020430, Sigma-Aldrich, St Louis, MI, USA), rabbit anti-AQP5 antibodies (1/1000, AB15858, Merck-Millipore, Burlington, MA, USA), mouse anti-β-actin (1/2000, #MAB1501, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: CLIP2: a novel functional player in AQP5 trafficking dynamics and implications for Sjögren’s disease

doi: 10.1186/s12964-025-02476-6

Figure Lengend Snippet: Computer docking model of the AQP5-CLIP2 complex. A Model of the complex between the AQP5 C-terminus (orange) and the two MTB-domains of CLIP2 (light cyan and teal respectively) generated by AlphaFold Multimer. AQP5 binds primarily to MTB1 in a manner that resembles how SLAIN2 (blue) interacts with CLIP1 MTB1 (grey) and how tubulin (magenta) interacts with CLIP1 MTB2 (white). B Zoom-in on the AQP5-CLIP2 interaction site. A stretch of acidic residues (EPDED) interacts with a highly conserved basic groove on MTB1 with hydrophobic residues on the proximal (I238, Y243) and distal side (W249) interacting with hydrophobic pockets on MTB1 and MTB2 respectively. C Crystal structure of human CLIP1 MTB1 in complex with a C-terminal peptide of SLAIN2 (PDB code 3RDV) and D NMR structure of human CLIP1 MTB2 in complex with the C-terminal tail of α-tubulin (PDB code 2E4H) showing a similar mode of interaction as in the predicted AQP5-CLIP2 complex

Article Snippet: The PVDF membranes were blocked for 1 h at RT with PBS containing 0.1% Tween 20 (PBS-T) and 5% skimmed milk, then incubated overnight at 4 °C with rabbit anti-CLIP2 antibody (1/1000, #25088-1-AP, Proteintech, Rosemont, IL, USA), rabbit anti-CLIP2 (1/1000, HPA020430, Sigma-Aldrich, St Louis, MI, USA), rabbit anti-AQP5 antibodies (1/1000, AB15858, Merck-Millipore, Burlington, MA, USA), mouse anti-β-actin (1/2000, #MAB1501, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Generated

Journal: Cell Communication and Signaling : CCS

Article Title: CLIP2: a novel functional player in AQP5 trafficking dynamics and implications for Sjögren’s disease

doi: 10.1186/s12964-025-02476-6

Figure Lengend Snippet: AQP5-CLIP2 interaction and co-localization. A-B PLA showing AQP5-CLIP2 complexes in NS-SV-AC cells ( A ) expressing SNAP-AQP5 and in hMSGB from SICCA-NS and SICCA-SD patients ( B ). Arrows indicate the localization of spots. Upper-right corner inserts show representative images used for the signal quantification (scale bar:30 μm). C Quantification of AQP5-CLIP2 complexes in SICCA-NS and SICCA-SD hMSGB. Results are expressed as the mean ± S.D. of AQP5-CLIP2 spots per cell ( n = 5). Data were analyzed using one-tailed Student’s t-test with Welch correction. D Localization of AQP5 and CLIP2 in hMSGB from SICCA-NS and SICCA-SD patients. AQP5 (AF594, red), CLIP2 (AF488, green) (scale bar: 25 μm). E Semi-quantification of AQP5 and CLIP2 localization in SICCA-NS and SICCA-SD hMSGB. Results are expressed as the median with the interquartile range of the labelled area for each protein relative to the entire hMSGB area ( n = 3). Data were analyzed using the one-tailed Mann-Whitney U test. F Co-localization of AQP5 and CLIP2 in SICCA-NS and SICCA-SD hMSGB. Arrows indicate the AQP5-CLIP2 co-localization (yellow area). Images are shown in their original version and modified forms (used for quantification) (scale bar: 50 μm). Negative control (NEG CTRL) was conducted in the absence of primary antibodies. G Semi-quantification of AQP5 and CLIP2 co-localization in SICCA-NS and SICCA-SD hMSGB. Results are expressed as the median with the interquartile range of the merged labelled area for both proteins relative to the entire hMSGB area ( n = 3). Data were analyzed using one-tailed Mann-Whitney U-test. One-tailed statistical tests were performed as values for SICCA-NS were not expected to be below SICCA-SD due to previously reported decreased expression of AQP5 in SICCA-SD hMSGB . C , E , G Statistical significance is indicated as *: p ≤ 0.05

Article Snippet: The PVDF membranes were blocked for 1 h at RT with PBS containing 0.1% Tween 20 (PBS-T) and 5% skimmed milk, then incubated overnight at 4 °C with rabbit anti-CLIP2 antibody (1/1000, #25088-1-AP, Proteintech, Rosemont, IL, USA), rabbit anti-CLIP2 (1/1000, HPA020430, Sigma-Aldrich, St Louis, MI, USA), rabbit anti-AQP5 antibodies (1/1000, AB15858, Merck-Millipore, Burlington, MA, USA), mouse anti-β-actin (1/2000, #MAB1501, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, One-tailed Test, MANN-WHITNEY, Modification, Negative Control

Journal: Cell Communication and Signaling : CCS

Article Title: CLIP2: a novel functional player in AQP5 trafficking dynamics and implications for Sjögren’s disease

doi: 10.1186/s12964-025-02476-6

Figure Lengend Snippet: PIP-CLIP2 interaction and co-localization. A-B PLA showing PIP-CLIP2 complexes in NS-SV-AC cells ( A ) and in hMSGB from SICCA-NS and SICCA-SD patients ( B ). Arrows indicate the localization of spots. Upper-right corner inserts show representative images used for the signal quantification (scale bar:30 μm). C Quantification of PIP-CLIP2 complexes in SICCA-NS and SICCA-SD hMSGB. Results are expressed as the mean ± S.D. of PIP-CLIP2 spots per cell ( n = 5). Data were analyzed using one-tailed Student’s t-test with Welch correction. D Colocalization of PIP and CLIP2 in hMSGB from SICCA-NS and SICCA-SD patients. PIP (AF594, red), CLIP2 (AF488, green). Images are shown in their original version and modified forms (used for quantification) (scale bar: 50 μm). Negative control (NEG CTRL) was conducted in the absence of primary antibodies. E Semi-quantification of PIP and CLIP2 localization in SICCA-NS and SICCA-SD hMSGB. Results are expressed as the median with the interquartile range of the labelled area for each protein relative to the entire hMSGB area ( n = 3). Data were analyzed using the one-tailed Mann-Whitney U test. C , E Statistical significance is indicated as *: p ≤ 0.05

Article Snippet: The PVDF membranes were blocked for 1 h at RT with PBS containing 0.1% Tween 20 (PBS-T) and 5% skimmed milk, then incubated overnight at 4 °C with rabbit anti-CLIP2 antibody (1/1000, #25088-1-AP, Proteintech, Rosemont, IL, USA), rabbit anti-CLIP2 (1/1000, HPA020430, Sigma-Aldrich, St Louis, MI, USA), rabbit anti-AQP5 antibodies (1/1000, AB15858, Merck-Millipore, Burlington, MA, USA), mouse anti-β-actin (1/2000, #MAB1501, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: One-tailed Test, Modification, Negative Control, MANN-WHITNEY

Journal: Cell Communication and Signaling : CCS

Article Title: CLIP2: a novel functional player in AQP5 trafficking dynamics and implications for Sjögren’s disease

doi: 10.1186/s12964-025-02476-6

Figure Lengend Snippet: EZRIN-CLIP2 interaction and co-localization. A-B PLA showing Ezrin-CLIP2 complexes in NS-SV-AC cells ( A ) and in hMSGB from SICCA-NS and SICCA-SD patients ( B ). Arrows indicate the localization of spots. Upper-right corner inserts show representative images used for the signal quantification (scale bar:30 μm). C Quantification of Ezrin-CLIP2 complexes in SICCA-NS and SICCA-SD hMSGB. Results are expressed as the mean ± S.D. of PIP-CLIP2 spots per cell ( n = 5). Data were analyzed using one-tailed Student’s t-test with Welch correction. D Colocalization of Ezrin and CLIP2 in hMSGB from SICCA-NS and SICCA-SD patients. Ezrin (AF594, red), CLIP2 (AF488, green). Images are shown in their original version and modified forms (used for quantification) (scale bar: 50 μm). Negative control (NEG CTRL) was conducted in the absence of primary antibodies. E Semi-quantification of Ezrin and CLIP2 localization in SICCA-NS and SICCA-SD hMSGB. Results are expressed as the median with the interquartile range of the labelled area for each protein relative to the entire hMSGB area ( n = 3). Data were analyzed using the one-tailed Mann-Whitney U test. C , E Statistical significance is indicated as *: p ≤ 0.05

Article Snippet: The PVDF membranes were blocked for 1 h at RT with PBS containing 0.1% Tween 20 (PBS-T) and 5% skimmed milk, then incubated overnight at 4 °C with rabbit anti-CLIP2 antibody (1/1000, #25088-1-AP, Proteintech, Rosemont, IL, USA), rabbit anti-CLIP2 (1/1000, HPA020430, Sigma-Aldrich, St Louis, MI, USA), rabbit anti-AQP5 antibodies (1/1000, AB15858, Merck-Millipore, Burlington, MA, USA), mouse anti-β-actin (1/2000, #MAB1501, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: One-tailed Test, Modification, Negative Control, MANN-WHITNEY

Journal: Cell Communication and Signaling : CCS

Article Title: CLIP2: a novel functional player in AQP5 trafficking dynamics and implications for Sjögren’s disease

doi: 10.1186/s12964-025-02476-6

Figure Lengend Snippet: PIP-Ezrin interaction and co-localization. A-B PLA showing PIP-CLIP2 complexes in NS-SV-AC cells ( A ) or in hMSGB from SICCA-NS and SICCA-SD patients ( B ). Arrows indicate the localization of spots. Upper-right corner inserts show representative images used for the signal quantification (scale bar:30 μm). C Quantification of PIP-CLIP2 complexes in SICCA-NS and SICCA-SD hMSGB. Results are expressed as the mean ± S.D. of PIP-CLIP2 spots per cell ( n = 5).Data were analyzed using one-tailed Student’s t-test with Welch correction. D Colocalization of PIP and CLIP2 in hMSGB from SICCA-NS and SICCA-SD patients. Ezrin (AF594, red), PIP (AF488, green). Images are shown in their original version and modified forms (used for quantification) (scale bar: 50 μm). Negative control (NEG CTRL) was conducted in the absence of primary antibodies. E Semi-quantification of Ezrin and CLIP2 localization in SICCA-NS and SICCA-SD hMSGB. Results are expressed as the median with the interquartile range of the labelled area for each protein relative to the entire hMSGB area ( n = 3). Data were analyzed using the one-tailed Mann-Whitney U test. C , E Statistical significance is indicated as *: p ≤ 0.05

Article Snippet: The PVDF membranes were blocked for 1 h at RT with PBS containing 0.1% Tween 20 (PBS-T) and 5% skimmed milk, then incubated overnight at 4 °C with rabbit anti-CLIP2 antibody (1/1000, #25088-1-AP, Proteintech, Rosemont, IL, USA), rabbit anti-CLIP2 (1/1000, HPA020430, Sigma-Aldrich, St Louis, MI, USA), rabbit anti-AQP5 antibodies (1/1000, AB15858, Merck-Millipore, Burlington, MA, USA), mouse anti-β-actin (1/2000, #MAB1501, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: One-tailed Test, Modification, Negative Control, MANN-WHITNEY

Journal: Current protocols in toxicology

Article Title: An Open-Globe Porcine Injury Platform for Assessing Therapeutics and Characterizing Biological Effects.

doi: 10.1002/cptx.98

Figure Lengend Snippet: Figure 5 Diagram of the benchtop injury device with pressure measurement system showing the solenoid puncture device (A), porcine eye in gelatin (B), catheter pressure transducer (PT; C), syringe pump (D), inline pressure transducer (E), and hydrostatic reservoir (F). The simplified diagrammatic version highlights the three-way valve positions (black = closed, blue = open).Valves identified as #1 and #2 are called out in the protocol. Further details behind the construction of the pressure measurement system and puncture device are described in Alternate Protocols 1 and 2. Figure adapted from Snider et al. (2020).

Article Snippet: Materials Prepared eye tissue in gelatin (see Basic Protocol 1) Phosphate-buffered saline (PBS; Gibco, cat. no. 10010-023) Pressure measurement system (Fig. 5): Syringe pump (KD Scientific, cat. no. 78-8100) 10-ml plastic Luer-Lok syringe (Becton-Dickinson, cat. no. 302995) Three-way valves (Cole-Parmer, cat. no. 30600-23) 23-G needle hub (McMaster-Carr, cat. no. 75165A684) Polyethylene tubing (Braintree Scientific, cat. no. PE-50, 100ft) Male × male Luer adapter (Cole-Parmer, cat. no. AO-45505-72) Saline bag/hydrostatic reservoir bag (Baxter, cat. no. 2B1322) 23-G needles (BD PrecisionGlide Needle, cat. no. 305143) Inline pressure transducer (MEMSCAP, AD Instruments, cat. no. SP 844) Inline pressure transducer domes (AD instruments, cat. no. MLA844) Pressure transducer interface cables (AD Instruments, cat. no. MLAC20) Catheter pressure transducer (Millar, cat. no. SPR-524) Catheter interface cable (AD Instruments, cat. no. AEC-10D) PowerLab data acquisition (DAQ) module (AD Instruments, cat. no. 16/35) Two Bridge Amp amplifiers (AD Instruments, cat. no. FE-221) Computer with LabChart 8 (AD Instruments) and Excel (Microsoft) Assemble pressure measurement system 1.

Techniques:

Journal: Frontiers in Medicine

Article Title: Identification and validation of novel risk genes for intervertebral disc disorder by integrating large-scale multi-omics analyses and experimental studies

doi: 10.3389/fmed.2025.1698050

Figure Lengend Snippet: DEGs and enrichment analysis of the three potential causal genes of IDD. (A) Volcano plot of the DEG analysis. Each dot represents a gene plotted according to the significance of the association measured as the −log 10 (FDR-adjusted p -value) (y-axis). The colors of the points are as follows: red for upregulated genes, blue for downregulated genes, and gray for non-differentially expressed genes (FDR-adjusted p ≥ 0.05). (B) Expression levels of potential causal genes for IDD in degenerative vs. non-degenerative groups, based on microassy data from clinical samples n = 5. Two-tailed t -test. * p < 0.05. (C) Significantly enriched pathways for the three potential causal genes ( TMEM190 , CILP2 and FOXO3 ) as determined by GSEA. Each line represents a pathway with significance defined by an FDR-adjusted p < 0.05. Yellow indicates upregulation (NES > 0), while green indicates downregulation (NES < 0).

Article Snippet: The membranes were blocked with a 5% skim milk solution at room temperature and then incubated overnight at 4°C with primary antibodies specific to TMEM190 (1:500; Invitrogen, PA5-70986), CILP2 (1:500; Proteintech, 11813-1-AP), and FOXO3 (1:1,000; Biotime Biotechnology, AF609-1).

Techniques: Expressing, Two Tailed Test

Journal: Frontiers in Medicine

Article Title: Identification and validation of novel risk genes for intervertebral disc disorder by integrating large-scale multi-omics analyses and experimental studies

doi: 10.3389/fmed.2025.1698050

Figure Lengend Snippet: Validation of risk genes in clinical IVD tissues. (A,B) Western-blot analysis of TMEM190, CILP2, and FOXO3 in control and severe IDD patients. n = 3. Data represent the mean (SD). Two tailed t -test. * p < 0.05.

Article Snippet: The membranes were blocked with a 5% skim milk solution at room temperature and then incubated overnight at 4°C with primary antibodies specific to TMEM190 (1:500; Invitrogen, PA5-70986), CILP2 (1:500; Proteintech, 11813-1-AP), and FOXO3 (1:1,000; Biotime Biotechnology, AF609-1).

Techniques: Biomarker Discovery, Western Blot, Control, Two Tailed Test

Journal: Frontiers in Medicine

Article Title: Identification and validation of novel risk genes for intervertebral disc disorder by integrating large-scale multi-omics analyses and experimental studies

doi: 10.3389/fmed.2025.1698050

Figure Lengend Snippet: Down-regulation of CILP2 alleviated IDD progression in mouse model of IDD. (A,B) Western-blot analysis of CILP2 in Sham, puncture (Punc) and knocking down (KD) IVD tissues in mouse model of IDD. n = 3. (C,D) Magnetic resonance imaging (MRI) and Pfirrmann grades of IVD in mice treated as in (A) . n = 3. (E,F) Hematoxylin and Eosin (H&E) staining and histological score assessment of IVD in mice treated as in (A) . n = 3. Scale bar = 200 μm. Data represent the mean (SD). One-way ANOVA. * p < 0.05.

Article Snippet: The membranes were blocked with a 5% skim milk solution at room temperature and then incubated overnight at 4°C with primary antibodies specific to TMEM190 (1:500; Invitrogen, PA5-70986), CILP2 (1:500; Proteintech, 11813-1-AP), and FOXO3 (1:1,000; Biotime Biotechnology, AF609-1).

Techniques: Western Blot, Magnetic Resonance Imaging, Staining

Journal: Ecotoxicology and environmental safety

Article Title: Exosomal circCLIP1 regulates PM 2.5 -induced airway obstruction via targeting SEPT10 in vitro.

doi: 10.1016/j.ecoenv.2023.114750

Figure Lengend Snippet: Fig. 2. Identification of loop structure and localization of circCLIP1. A. Sanger sequencing confirmed the head-to-tail splicing of circCLIP1. B. Expression of circular and linear CLIP1 after amplified by random hexamer or Oligo (dT)18 primer was determined by RT-qPCR. C. Expression of back-spliced and canonical form of CLIP1 in cDNA and gDNA was measured by agarose gel electrophoresis assay. D. Expression of circular and linear CLIP1 with/without RNase R treatment was assessed by RT-qPCR. E. Subcellular localization of circCLIP1 was detected by fluorescence in situ hybridization. Scale bar, 50 µm. F. Expression of circCLIP1 in culture medium of PM2.5-treated HBE cells treated with DMSO or GW4869 was determined by RT-qPCR. Data were represented as means ± SD (n = 3). Statistical significance was assessed using two-tailed Student’s t test, * P < 0.05.

Article Snippet: The primary antibodies included Alix (92880, CST, USA), CD63 (ab134045, Abcam, USA), MUC5AC (ab198294, Abcam, USA), CLCA1 (ab180851, Abcam, USA), β-actin (66009–1-Ig, Proteintech, China), GAPDH (60004–1-Ig, Proteintech, China), SM-MHC (ab53219, Abcam, USA), α-SMA (19245, Cell Signaling Technology, USA), RhoA (2117, Cell Signaling Technology, USA), SEPT10 (A304–915A, ThermoFisher, USA), DKC1 (ab156877, Abcam, USA) and CLIP1 (23839–1-AP, Proteintech, China).

Techniques: Sequencing, Expressing, Amplification, Random Hexamer, Quantitative RT-PCR, Agarose Gel Electrophoresis, Fluorescence, In Situ Hybridization, Two Tailed Test