MA5-12104

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of Cytokeratin 18 using Cytokeratin 18 Monoclonal Antibody (Product # MA5-12104) on HT29 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell and tissue extracts of MCF7 (Lane 1), Mouse prostrate tissue (Lane 2), Hep G2 (Lane 3) and Mouse liver tissue (Lane 4). The blots were probed with Anti-Cytokeratin 18 Mouse Monoclonal Antibody (Product # MA5-12104, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A ~ 50 kDa band corresponding to Cytokeratin 18 was observed across cell line and tissue extracts tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of MCF7 (Lane 1), A549 (Lane 2), A-375 (Lane 3) and A-431 (Lane 4). The blot was probed with anti-Cytokeratin 18 Mouse Monoclonal Antibody (Product # MA5-12104, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 50 kDa band corresponding to Cytokeratin 18 was observed in MCF7 and A549, and not observed in other cell lines which are documented to be Cytokeratin 18 negative.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Stained image of a cross-section of the tubular liver tissue perpendicular to the main blood vessel. ( a ) hematoxylin and eosin (HE) staining image. ( b ) immunohistochemistry (IHC) image (CD31). Asterisks indicate the main blood vessels. Blue arrowheads indicate capillaries. Red arrowheads indicate branches from main vessels to capillaries. ( c ) Immunofluorescence image (CK18 and CD31). ( d ) Schematic image of the tubular liver tissue.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Primary bEEC culture and identification. (A) Morphological observation of bEECs by ordinary optical microscopy (magnification, x200). (B) Primary bEECs were positive for the epithelial-specific marker CK-18 (red). (C) Nuclei were stained with DAPI (blue). (D) Merge of CK-18 and DAPI (magnification, x400). (E) The CK-18 + cells were counted using ImageJ. bEEC, bovine endometrial epithelial cell; CK-18, cytokeratin-18; IF, immunofluorescence.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5. DSP -kd disrupts cell-cell junctions and cytoskeletal organization. CRISPRi-iAT2s were transduced with DSP gRNA and treated without (-dox) or with (+dox) doxycycline. ( A ) Immunofluorescence staining of desmoplakin (DSP). DSP, red; nuclei, blue; scale bar, 10 mum. ( B ) Flow cytometry analysis of DSP expression, represented by mean fluorescence intensity (MFI). n = 3 experimental replicates of independent wells of a differentiation; error bars represent SD. Statistical significance was determined by unpaired, two-tailed Student's t test; *** P < 0.001. ( C ) Transmission electron microscopy images showing tight junctions (TJ) and desmosomes (De); scale bar, 200 nm. ( D ) Violin plots showing module scores for KEGG (Kyoto Encyclopedia of Genes and Genomes) tight junction, cell adhesion molecules, and gap junction. ( E ) Dot plots showing expression of genes associated with desmosome, ( F ) adherens junction, and ( G ) TJ. Genes that were not expressed by iAT2s were excluded from dot plots. ( H ) Immunofluorescence staining of claudin-4, ZO-1, and E-cadherin. ( I ) Immunofluorescence staining of keratin-18 and alpha-tubulin. Arrows indicate disorganized filaments or tubules. Protein of interest, red; nuclei, blue; scale bar, 10 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 3. GRHL3 is required to activate SE identity genes through opening their chromatin accessibilities. ( A ) Wild-type (WT) and GRHL3 -knockout hESCs during SE differentiation. Top: Phase contrast images. Bottom: Immunofluorescence staining of GRHL3 (green) and KRT18 (red). Scale bars, 100 mum. ( B ) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of representative genes in wild-type and GRHL3 -knockout hESCs after 2 days of differentiation. qRT-PCR values were normalized to the values in wild-type group. Values are presented as means +- SD ( n = 3 biological replicates; * P < 0.05; ** P < 0.01; *** P < 0.001; t test). ( C ) Scatterplot of differential accessibility in wild-type versus GRHL3 -knockout (KO) hESCs after 2 days of differentiation. Sites identified as significantly differentially bound [log 2 fold change > 1 or < -1 and false discovery rate (FDR) < 0.05] are shown in color (red, peaks increased; blue, peaks decreased). ( D ) Metaplots of average ATAC-seq density around the SE identity genes in wild-type and GRHL3 -knockout hESCs after 2 days of differentiation. ( E ) Gene set enrichment analysis of the SE identity gene set in the gene expression matrix of wild-type and GRHL3 -knockout hESCs after 2 days of differentiation. NES, normalized enrichment score. ( F ) Enrichment of transcription factor motifs identified by HOMER at GRHL3 peaks. ( G ) Heatmaps of the binding signals of TFAP2C and GRHL2 at the center of GRHL3 peaks. ( H

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4. GRHL3 induces SE commitment. ( A ) Schematic diagram of establishment of a TetO-GRHL3 doxycycline-inducible expression system in hESC. Cells were induced with doxycycline for 7 days. TRE, tetracycline response element; rtTA, reverse tetracycline transactivator. ( B ) Immunofluorescence staining of GRHL3 (green) and KRT18 (red) in TetO-GRHL3 + and TetO-GRHL3 - cells. Left: Phase contrast images. Scale bars, 100 mum. ( C ) qRT-PCR analysis of representative genes in TetO-GRHL3 + and TetO-GRHL3 - cells. qRT-PCR values were normalized to the values in TetO-GRHL3 - cells. Values are presented as means +- SD ( n = 3 biological replicates; ** P < 0.01; *** P < 0.001; t test). ( D ) Heatmap of germ layer-specific gene expression levels in TetO-GRHL3 + and TetO-GRHL3 - cells. ( E ) PCA of RNA-seq data of TetO-GRHL3 + cells and hESCs at each time points during SE differentiation. ( F ) Genome browser tracks comparing ATAC-seq signal at KRT8/18, KRT7, and TFAP2C loci in TetO-GRHL3 + and TetO-GRHL3 - cells. ( G ) Left: Scatterplot of differential accessibility in TetO-GRHL3 + versus TetO-GRHL3 - cells. Sites identified as significantly differentially bound are shown in color (red, peaks increased; blue, peaks decreased). Right: Transcription factor motif enrichment in the regions of differential accessibility in TetO-GRHL3 + versus TetO-GRHL3 - cells. TEAD, TEA domain transcription factor.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Inducible EGFP-expressing and CTEN-expressing RWPE-1 cells perform similar phenotypes in the absence of doxycycline in 3D culture. Inducible EGFP-expressing (aON-EGFP) or CTEN-expressing (aON-CTEN) RWPE-1 cells were incubated in 3D culture system for 9 days without doxycycline induction. ( a ) The appearance of acinar structures was observed and classified to three types (normal, notched and deformed). The numbers of the three-type acini in each group were counted and presented as a percentage of the whole (upper panel). Data were expressed as mean +- SD of three independent experiments. More than 50 acini were evaluated within each sample (Student's t test; no significant difference between aON-EGFP and aON-CTEN in each phenotype). The representative images of acinar structure were also shown in the lower panel. Scale bar: 100 mum. ( b ) aON-EGFP and aON-CTEN RWPE-1 cells grown in 3D culture were collected at the indicated time and the protein levels were examined by Western analyses using the indicated antibodies. alpha-tubulin was used as a loading control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 10 Cytoskeletal filaments decorate the CCV. HeLa cells were infected with C. burnetii at an MOI of 50. At 72 h post-infection, cells were fixed and stained with Phallotoxin-647 for actin, or with tubulin- and cytokeratin 18-specific antibodies by indirect immunofluorescence (green). Nuclei and bacterial DNA were stained with DAPI (blue). Cells were visualized using LSM. N: Nucleus. CCV: C. burnetii -containing vacuole.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 11 Cytoskeletal filaments are not modified by AnkF expression. HeLa cells were transiently transfected with pGFP alone or with pEGFP-AnkF (green). 24 h post-transfection, cells were fixed and stained with Phalloidin-Alexa647 for actin, or with tubulin- and cytokeratin 18-specific antibodies by indirect immunofluorescence (red). Nuclei and bacterial DNA were stained with DAPI (blue). Cells were visualized using LSM. Representative images from two independent experiments are shown.