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