Journal: CNS Neuroscience & Therapeutics

Article Title: MGCD 0103, a selective histone deacetylase inhibitor, coameliorates oligomeric Aβ 25‐35 ‐induced anxiety and cognitive deficits in a mouse model

doi: 10.1111/cns.13029

Figure Lengend Snippet: List of primary antibodies in immunohistochemistry and Western blot

Article Snippet: pCDK5 , Mouse , Santa Cruz, Dallas, TX, USA , 1:1000 , .

Techniques: Immunohistochemistry, Western Blot

Journal: Nature communications

Article Title: Cdk5 controls lymphatic vessel development and function by phosphorylation of Foxc2.

doi: 10.1038/ncomms8274

Figure Lengend Snippet: Figure 1 | Endothelial cell-specific Cdk5 knockout mice. (a) Impaired survival of Cdk5fl/flTie2Cre mice. The Kaplan–Meier plot indicates that 40% Cdk5fl/flTie2Cre mice died during the first 2 days, 75% during the first 30 days after birth; 352 mice; 54 Cdk5fl/flTie2Cre mice. (b) Reduced size of Cdk5fl/flTie2Cre mice (d11). Scale bar, 2 cm. (c) Reduced body weight of Cdk5fl/flTie2Cre mice. t-test, *Pr0.05, s.e.m.; nZ4 per age and genotype. (d) Intestinal bleeding of Cdk5fl/flTie2Cre mice (d20). Scale bar, 1 cm. (e) Embryonic lethality of Cdk5fl/flTie2Cre embryos. Percent of living Cdk5fl/flTie2Cre embryos at indicated stages. (f–h) Blood-filled leaky superficial capillaries, bleeding and edema formation in EC-specific Cdk5 knockout embryos. (f) E15.5 and E16.5 Cdk5fl/flTie2Cre embryos. Scale bar, 2 mm. (g) E16.5 Cdk5fl/flVECCre embryos. Scale bar, 2 mm. (h) E15.5 Cdk5D/flTie2Cre embryos. Scale bar, 2 mm. (i) Decreased Cdk5 levels in LECs (29%) and BECs (41%) of E16.5 Cdk5fl/flTie2Cre embryos. Each: t-test, *Pr0.001, s.e.m.; n ¼ 8 per genotype. (j) Decreased Cdk5 levels in LECs (7.8%) and BECs (6.4%) of E15.5 Cdk5D/flTie2Cre embryos. Each: t-test, *Pr0.001, s.e.m.; n ¼ 3 per genotype.

Article Snippet: Plasmids: Cdk5 (addgene 1871), p35 (addgene 1347), Foxc2 (ref. 39), Foxc2-mutA8 and Foxc2-mutD219–366 (ref. 31).

Techniques: Knock-Out

Journal: Nature communications

Article Title: Cdk5 controls lymphatic vessel development and function by phosphorylation of Foxc2.

doi: 10.1038/ncomms8274

Figure Lengend Snippet: Figure 3 | Defective lymphatic valve formation in EC-specific Cdk5 knockout embryos. (a) Cdk5 expression in lymphatic valves. Whole-mount stainings of E18.5 mesenteric vessels show co-localization of Cdk5 (green) and Prox1 (red) (arrowheads). n ¼ 3. Scale bar, 50 mm. (b–e) Cdk5 controls lymphatic valve formation. Stainings of (b) skin and (c) mesenteric vessels of E16.5 embryos for Prox1 (green), Foxc2 (blue) and a-SMA (red). Valves are indicated by asterisks. n ¼ 9 per genotype. Scale bar, 50 mm. (d,e) Quantification of valves in E16.5 embryos. n ¼ 9 per genotype. (d) skin; t-test, *Pr0.001, s.e.m. (e) mesenteric vessels; t-test, *Pr0.001, s.e.m. (f–i) Cdk5 controls lymphatic valve maturation. Stainings of (f) skin and (g) mesenteric vessels of E18.5 embryos for Prox1 (green), Foxc2 (blue) and a-SMA (red). Valves are indicated by asterisks. n ¼ 5 per genotype. Scale bar, 50 mm. (h,i) Quantification of valves in E18.5 embryos. (h) Skin; t-test, *Pr0.05, s.e.m. (i) Mesenteric vessels; t-test, *Pr0.05, s.e.m; (h,i) n ¼ 5 per genotype.

Article Snippet: Plasmids: Cdk5 (addgene 1871), p35 (addgene 1347), Foxc2 (ref. 39), Foxc2-mutA8 and Foxc2-mutD219–366 (ref. 31).

Techniques: Knock-Out, Expressing

Journal: Nature communications

Article Title: Cdk5 controls lymphatic vessel development and function by phosphorylation of Foxc2.

doi: 10.1038/ncomms8274

Figure Lengend Snippet: Figure 4 | Impaired lymphovenous valve formation in Cdk5 knockout embryos. Sagittal views of control and Cdk5fl/flTie2Cre embryos (E12.5) whole- mount immunostained for CD31 (green), VEGFR3 (blue) and Prox1 (red) are shown (left panels). Two contact sites between pTD and CV that express high levels of Prox1 indicate lymphovenous valves (arrowheads). Scale bar, 500 mm. Individual optical sections (right panels) through the contact area of pTD and CV are shown. In the control, two areas with a double layer of endothelial cells that express high levels of Prox1 indicate lymphovenous valves (arrowheads). Scale bar, 100 mm. Control: n ¼ 4. Three out of five analysed Cdk5fl/flTie2Cre embryos showed defects in lymphovenous valve formation.

Article Snippet: Plasmids: Cdk5 (addgene 1871), p35 (addgene 1347), Foxc2 (ref. 39), Foxc2-mutA8 and Foxc2-mutD219–366 (ref. 31).

Techniques: Knock-Out, Control

Journal: Nature communications

Article Title: Cdk5 controls lymphatic vessel development and function by phosphorylation of Foxc2.

doi: 10.1038/ncomms8274

Figure Lengend Snippet: Figure 5 | Cdk5 is not essential for valve maintenance. (a) Normal lymphatic vessels in postnatally induced endothelial Cdk5 knockout mice. Mesentery of d10 control and Cdk5fl/flVECCre pups treated with tamoxifen (d1–d3). Lymphatic vessel (L), artery (A) and vein (V) are indicated. n ¼ 2 per genotype. Scale bar, 2 mm. (b) Normal lymphatic valves in postnatally induced endothelial Cdk5 knockout mice. Staining of mesentery of d10 control and Cdk5fl/flVECCre pups for Prox1 (green) and a-SMA (red). n ¼ 2 per genotype. Scale bar, 50 mm.

Article Snippet: Plasmids: Cdk5 (addgene 1871), p35 (addgene 1347), Foxc2 (ref. 39), Foxc2-mutA8 and Foxc2-mutD219–366 (ref. 31).

Techniques: Knock-Out, Control, Staining

Journal: Nature communications

Article Title: Cdk5 controls lymphatic vessel development and function by phosphorylation of Foxc2.

doi: 10.1038/ncomms8274

Figure Lengend Snippet: Figure 6 | Phenotypes of endothelial Cdk5 and Foxc2 knockout embryos are similar. (a) Blood-filled superficial capillaries, bleeding and edema formation in Foxc2 knockout embryos (Foxc2 / , E13.5) are shown. Representative images of Foxc2 þ / þ (n ¼ 1), Foxc2 þ / (n ¼ 6) and Foxc2 / (n ¼ 6 with n ¼ 4 with obvious phenotype) littermates are shown. Scale bar, 2 mm at left panels. Scale bar, 1 mm at right panels. (b) Immunostainings reveal blood-filled lymphatic vessels in Foxc2 / embryos. Lyve1 (red) indicates lymphatic vessels. Blood cells are stained by Ter119 (green). Scale bar, 10 mm.

Article Snippet: Plasmids: Cdk5 (addgene 1871), p35 (addgene 1347), Foxc2 (ref. 39), Foxc2-mutA8 and Foxc2-mutD219–366 (ref. 31).

Techniques: Knock-Out, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Role of cyclin-dependent kinase 5 in early brain injury following experimental subarachnoid hemorrhage

doi: 10.3892/etm.2021.11070

Figure Lengend Snippet: Expression levels of Cdk5, Cdk5-pTyr15 and p25 over time following SAH. (A) Representative autoradiograms of the expression levels of Cdk5, Cdk5-pTyr15 and p25 in the temporal cortex following SAH. Quantitative analysis of the western blotting results; (B) Cdk5 protein levels significantly increased at 12 h and on day 1 after SAH; (C) Cdk5-pTyr15 protein levels increased at 6 and 12 h and on day 1 after SAH; and (D) p25 protein levels increased on days 1 and 3 after SAH. (E) Quantified ratio of Cdk5-pTyr15/Cdk5 expression. * P<0.05, ** P<0.01 compared with the sham group. Cdk5, cyclin-dependent kinase 5; Cdk5-pTyr15, Cdk5 phosphorylated at Tyr15; SAH, subarachnoid hemorrhage.

Article Snippet: In total, 20 µg proteins were then separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane, which was blocked for 30 min in 5% non-fat milk in 1X TBS-0.1% Tween at 37˚C and then incubated with primary antibodies at 37˚C for Cdk5 (1:1,000, cat. no. ab40773; Abcam), Cdk5-pTyr15 (1:1,000; cat. no. AP55874PU-N; OriGene Technologies, Inc.), p25 (1:1,000; cat. no. ab125653; Abcam) and β-actin (1:1,000; cat. no. ab8226; Abcam) overnight.

Techniques: Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Role of cyclin-dependent kinase 5 in early brain injury following experimental subarachnoid hemorrhage

doi: 10.3892/etm.2021.11070

Figure Lengend Snippet: Double-immunofluorescence staining of Cdk5 after SAH. Cdk5 (green), NeuN (red) and GFAP (red) in the sham and day 1 post-SAH groups; nuclei were counterstained with DAPI (blue). Overlapping images show that Cdk5 was expressed in neurons and astrocytes in both the sham and SAH groups. Cdk5 was mainly expressed in the neuronal cytoplasm in the sham group; however, Cdk5 translocated to the nucleus after SAH (white arrows). Moreover, enhanced Cdk5 immunoreactivity was detected in the astrocytes of the SAH group compared with those of the sham group after SAH (scale bars, 20 µm). Cdk5, cyclin-dependent kinase 5; NeuN, neuronal nuclei; GFAP, glial fibrillary acidic protein; SAH, subarachnoid hemorrhage.

Article Snippet: In total, 20 µg proteins were then separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane, which was blocked for 30 min in 5% non-fat milk in 1X TBS-0.1% Tween at 37˚C and then incubated with primary antibodies at 37˚C for Cdk5 (1:1,000, cat. no. ab40773; Abcam), Cdk5-pTyr15 (1:1,000; cat. no. AP55874PU-N; OriGene Technologies, Inc.), p25 (1:1,000; cat. no. ab125653; Abcam) and β-actin (1:1,000; cat. no. ab8226; Abcam) overnight.

Techniques: Double Immunofluorescence Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Role of cyclin-dependent kinase 5 in early brain injury following experimental subarachnoid hemorrhage

doi: 10.3892/etm.2021.11070

Figure Lengend Snippet: Effects of roscovitine on Cdk5-pTyr15 expression. (A) Cdk5-pTyr15 expression, as demonstrated by western blotting. (B) Quantitative western blotting results for Cdk5-pTyr15/β-actin expression. (C) Quantitative western blotting results for Cdk5-pTyr15/Cdk5. Roscovitine (100 µg) treatment significantly inhibited SAH-induced Cdk5-pTyr15 upregulation. ** P<0.01 compared with the sham + vehicle group; ## P<0.01 compared with the SAH + vehicle group. Cdk5-pTyr15. SAH, subarachnoid hemorrhage; Cdk5-pTyr15, Cdk5 phosphorylated at Tyr15.

Article Snippet: In total, 20 µg proteins were then separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane, which was blocked for 30 min in 5% non-fat milk in 1X TBS-0.1% Tween at 37˚C and then incubated with primary antibodies at 37˚C for Cdk5 (1:1,000, cat. no. ab40773; Abcam), Cdk5-pTyr15 (1:1,000; cat. no. AP55874PU-N; OriGene Technologies, Inc.), p25 (1:1,000; cat. no. ab125653; Abcam) and β-actin (1:1,000; cat. no. ab8226; Abcam) overnight.

Techniques: Expressing, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Phosphorylation of PBK at Thr9 by CDK5 correlates with invasion of prolactinomas

doi: 10.1111/cns.14629

Figure Lengend Snippet: CDK5 promotes cell proliferation and EMT progression in GH3 cells. (A, B) Growth curves of GH3 cells treated with roscovitine and transfected with siCDK5‐2, siCDK5‐3, and CDK5. (C, D) EdU assay for detecting cell proliferation. E and F Wound healing assay for detecting cell invasion. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. The bar represents the mean ± SD.

Article Snippet: The CDK5 siRNA (SR507441) construct was procured from OriGene Technologies located in Rockville, MD, USA.

Techniques: Transfection, EdU Assay, Wound Healing Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Phosphorylation of PBK at Thr9 by CDK5 correlates with invasion of prolactinomas

doi: 10.1111/cns.14629

Figure Lengend Snippet: Phosphorylation of PBK at Thr9 by CDK5 promotes cell proliferation and EMT progression. (A) GH3 cells treated with roscovitine and transfected with siCDK5‐2, siCDK5‐3, and CDK5. Western blot analysis of pPBK‐T9, PBK, CDK5, and EMT expression. (B) Co‐IP to detect the interaction between CDK5 and PBK. (C) I n vitro kinase assay was performed to test whether CDK5 phosphorylates PBK at Thr9. (D) The phosphorylation site of PBK at Thr9 was analyzed by mass spectrometry. (E) Growth curves of GH3 cells transfected with PBK and PBK‐T9F. (F) EdU assay for detecting GH3 cells transfected with PBK and PBK‐T9F. (G) Wound healing assay for detecting cell invasion. (H) Western blot analysis of pPBK‐T9, PBK, CDK5, and EMT expression after GH3 cells were transfected with PBK and PBK‐T9F. (I) GH3 cells were transfected with PBK and PBK‐T9F, and the expression of MAPK signaling pathway‐related proteins was determined . (J) Western blot analysis was performed to test whether PBK reversed the effect of siCDK5 on EMT progression. β‐Actin served as a loading control. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. The bar represents the mean ± SD.

Article Snippet: The CDK5 siRNA (SR507441) construct was procured from OriGene Technologies located in Rockville, MD, USA.

Techniques: Phospho-proteomics, Transfection, Western Blot, Expressing, Co-Immunoprecipitation Assay, Kinase Assay, Mass Spectrometry, EdU Assay, Wound Healing Assay, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Phosphorylation of PBK at Thr9 by CDK5 correlates with invasion of prolactinomas

doi: 10.1111/cns.14629

Figure Lengend Snippet: Phosphorylation of PBK at Thr9 by CDK5 enhances the stability of PBK. (A) HEK293T cells were transfected with the indicated plasmids, and cell extracts were subjected to IP with an anti‐FLAG antibody. Ubiquitinated PBK was detected by immunoblotting. (B, C) HEK293T cells were transfected with the indicated plasmids and then treated with CHX (100 μg/mL) to prevent new protein synthesis. The time‐dependent stability of PBK was detected by Western blot. (D) Validation of proteasome degradation pathways. β‐Actin served as a loading control.

Article Snippet: The CDK5 siRNA (SR507441) construct was procured from OriGene Technologies located in Rockville, MD, USA.

Techniques: Phospho-proteomics, Transfection, Western Blot, Biomarker Discovery, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Phosphorylation of PBK at Thr9 by CDK5 correlates with invasion of prolactinomas

doi: 10.1111/cns.14629

Figure Lengend Snippet: Phosphorylation of PBK at Thr9 by CDK5 promotes prolactinoma growth in vivo. (A) Prolactinomas in F344 rats were induced by E2 administration. MRI shows lesions located on the seller's floor (left). The pituitary tumor volume was measured by MRI (right). (B) IHC analysis of pPBK‐T9, E‐cadherin, and N‐cadherin expression in tumor sections from prolactinomas in F344 cells. Magnification, 400×. (C) Tumors dissected from the NC, PBK, PBK‐T9F, roscovitine, and roscovitine+PBK groups are shown (left). Growth curve of tumor size and average tumor weight (right). (D) Western blot analysis of the expression of the indicated markers in tumor sections from mice in the NC, PBK, PBK‐T9F, roscovitine, and roscovitine+PBK groups. β‐Actin served as a loading control. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The bar represents the mean ± SD.

Article Snippet: The CDK5 siRNA (SR507441) construct was procured from OriGene Technologies located in Rockville, MD, USA.

Techniques: Phospho-proteomics, In Vivo, Expressing, Western Blot, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Phosphorylation of PBK at Thr9 by CDK5 correlates with invasion of prolactinomas

doi: 10.1111/cns.14629

Figure Lengend Snippet: pPBK‐T9 is highly expressed in invasive PitNETs. (A) IHC analysis of pPBK‐T9, PBK, CDK5, and p ‐CDK5 expression in PitNET tissue samples. Magnification, 400×. (B) IHC score of pPBK‐T9 in different Knop grade PitNETs. (C, D) IHC score of pPBK‐T9 and PBK in prolactinomas. ***, p < 0.001. The bar represents the mean ± SD.

Article Snippet: The CDK5 siRNA (SR507441) construct was procured from OriGene Technologies located in Rockville, MD, USA.

Techniques: Expressing

Journal: CNS Neuroscience & Therapeutics

Article Title: Phosphorylation of PBK at Thr9 by CDK5 correlates with invasion of prolactinomas

doi: 10.1111/cns.14629

Figure Lengend Snippet: Schematic representation showing the proposed mechanisms through which the Phosphorylation of PBK at Thr9 by CDK5 promotes the prolactinoma progression.

Article Snippet: The CDK5 siRNA (SR507441) construct was procured from OriGene Technologies located in Rockville, MD, USA.

Techniques: Phospho-proteomics

Journal: Nature communications

Article Title: A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer's disease.

doi: 10.1038/s41467-023-42704-6

Figure Lengend Snippet: Fig. 5 | REST suppresses the tau kinases CDK5 and GSK3β. a, b Loss of REST in excitatory neurons increases CDK5 expression in cortex and hippocampus. aImmunolabelingforCDK5(green)andthe neuronalmarkerMAP2(magenta)in CA1 neurons of the hippocampus in 9-month-old 3xTg and 3xTg;cKO mice. b Quantification of CDK5 immunofluorescence intensity in the hippocampus and cortex of 9-month-old 3xTg (n = 4) and 3xTg;cKO (n = 4) mice. c, d Loss of a single REST allele increases CDK5 expression. c Immunolabeling for CDK5 (green) and MAP2 (magenta) in 29-month-old 3xTg and 3xTg;GT (heterozygous REST null) mice. d Quantification of CDK5 immunofluorescence intensity in 28–29-month-old 3xTg (n = 6) and 3xTg;GT (n = 6) mice. e, f Loss of REST in excitatory neurons increases GSKβ expression in cortex and hippocampus. e Immunolabeling for GSK3β (green) and MAP2 (magenta) in hippocampal CA1 neurons in 9-month-old 3xTg and 3xTg;cKO mice. f Quantification of GSK3β immunofluorescenceintensityin9-month-old3xTg(n = 4)and3xTg;cKO (n = 4) mice. g, h Loss of a single REST allele increases GSK3β expression.

Article Snippet: Additional primary antibodies were as follows: anti-human Aβ rabbit monoclonal IgG antibody (Cell Signaling, Cat. No. 8243); antihuman APP mouse monoclonal IgG antibody (clone 6E10; Covance, Catalog No. SIG-39320); anti-actin mouse monoclonal IgG antibody (clone ACTN05 (C4); ThermoFisher Scientific, CatalogNo.MA5-11869); anti-NeuN mouse monoclonal IgG antibody (clone A60, Millipore, MAB377); anti-MAP2 goat polyclonal IgG antibody (PhosphoSolutions, Catalog. No. 1099-MAP2); anti-CDK5 mouse monoclonal IgG antibody (clone 4E4; Novus Bio, Catalog No. NBP2-37602); anti-GSK3β mouse monoclonal IgG antibody (clone D5C5Z; Novus Bio, catalog No. NBP147470S); anti-PS1 C-terminal (CTF) rabbit monoclonal IgG antibody (clone EP2000Y; Abcam, Catalog No. ab76083); anti-PS1 N-terminal (NTF) rabbit polyclonal IgG antibody (231-f; made in the Yankner lab); anti-Nicastrin mouse monoclonal IgG antibody (clone 9C3; Biolegend, Catalog No. 852301); anti-Nicastrin rabbit polyclonal IgG antibody (Sigma Millipore, Catalog No. N1660); anti-PEN2 rabbit polyclonal IgG antibody (ProScience, Catalog No.3981); anti-PEN2 rabbit monoclonal IgG antibody (clone EPR9200; Abcam, Catalog No. ab154830); antiPEN2 rabbit polyclonal IgG (ProScience, Catalog No. 3981); antiTransferrin receptor mouse monoclonal IgG antibody (clone H68.4; ThermoFisher Scientific, Catalog No. 13-6800); anti-BiP/GRP78 mouse monoclonal IgG (clone C38; ThermoFisher Scientific, clone C38, CatalogNo.

Techniques: Expressing, Immunolabeling

Journal: The Journal of Neuroscience

Article Title: Dynamic Partitioning of Synaptic Vesicle Pools by the SNARE-Binding Protein Tomosyn

doi: 10.1523/jneurosci.1297-16.2016

Figure Lengend Snippet: Figure3. Tomo1servesascatalyticsubstrateforneuralactivity-sensitiveCdk5.A1,RepresentativeimmunofluorescentimagesofSynapsin1(cyan,top)andphosphorylatedCdk5(pCdk5;heat scale,bottom)instraightenedaxonsegmentsfromneuronstreated(30min)withCSA(50M),Rosco(100M),orcontrol(DMSO,0.5%).A2,AveragedpCdk5immunofluorescenceintensityrelative to vehicle control. A3, Cumulative frequency of pCdk5 intensity across boutons shows that inhibition of Cdk5 by Rosco significantly reduces pCdk5 immunoreactivity, whereas CSA treatment enhances pCdk5 relative to control. B1–B3, pCdk5 immunofluorescence images comparing chronic silencing of neural activity by TTX (24 h, 1 M) with control. Averaged pCdk5 intensity (B2) and cumulativefrequencyofpCdk5(B3)intensitydemonstratethatchronicdampeningofneuralactivitysignificantlyreducesCdk5activation.A,B,Analysiswasperformedon45images/preparation per condition and repeated on 3 neuronal preparations. C, Immunoblots showing coprecipitation of Tomo1 and Cdk5 from cultured hippocampal neuron lysates regardless of primary target of IP (Tomo1 or Cdk5). D, In vitro phosphorylation of affinity-purified Tomo1 by Cdk5/p25 as measured by 32P labeling. 32P-radioactive signal (top) and anti-Tomo1 immunoreactivity (bottom) on Western blot corresponding to the following conditions: absence of Cdk5/p25 kinase (H2O), Cdk5/p25 kinase (Cdk5), and Cdk5/p25 1 mM Rosco (Cdk5 Rosco). *p 0.05.

Article Snippet: For in vitro Cdk5/p25 phosphorylation reactions, 11.5 g of mTomo1 on streptavidin beads was rinsed in phosphorylation buffer containing the following (in mM): 25 MOPS, pH 7.2, 12.5 -glycerol phosphate, 25 MgCl2, 5 EGTA, 2 EDTA, 0.25 DTT, and 250 g of active Cdk5/p25 kinase (Signal Chem, C33–10G).

Techniques: Control, Inhibition, Immunofluorescence, Activity Assay, Western Blot, Cell Culture, In Vitro, Phospho-proteomics, Affinity Purification, Labeling