Journal: The FASEB Journal

Article Title: Full‐Length Cryptochrome 1 in the Outer Segments of the Retinal Blue Cone Photoreceptors in Humans and Great Apes Suggests a Role Beyond Transcriptional Repression

doi: 10.1096/fj.202402614r

Figure Lengend Snippet: FIGURE 1 | Full-length CRY1 label (detected with the gp-α-CRY1 serum) in the SWS1 (blue) cones of human, gorilla, bonobo, and orangutan retina. Transverse section (top row) and flatmounted piece (second row) of human retina, flatmounted retinal pieces of the other species with focus on the photoreceptor layer. Left column: CRY1 immunofluorescence (green). Middle column: SWS1 opsin immunofluorescence located in the blue cone outer segments and to a lesser extent in the larger blue cone inner segments (magenta). Right column: Merged images, showing that CRY1 and SWS1 opsin colocalize in all blue cone outer segments. The bonobo field is from the central retina, the gorilla and orangutan fields are from the pe- ripheral retina, and the human field is from an unknown location. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, IS, photoreceptor outer and inner segments, respectively. Scale bar in the top left image is 50 μm and applies to all images.

Article Snippet: Antibody name Species and antibody clonality Immunization peptide Dilution Source gp- α- CRY1 Guinea pig polyclonal RPNPEEETQSVGPKVQRQST hCRY1 sequence 566–585 IHC: 1:100 WB: 1:500 Genovac GmbH, Freiburg, Germany [32] rb- α- CRY1 PA1- 527 Rabbit polyclonal QSVGPKVQRQSSN hCRY1 sequence 574–586 IHC: 1:500 WB: 1:1000 Thermo Fisher Scientific, Waltham, MA, USA rt- α- CRY1 3E12 Rat monoclonal RPNPEEETQSVGPKVQRQST hCRY1 sequence 566–585 IHC: undiluted Monoclonal Antibody Core Facility, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany [33] rt- α- CRY1 17A2 Rat monoclonal TTPVSDDHDEKYG hCRY1 sequence 179–191 IHC: 1:2 WB 1:1000 This study; Monoclonal Antibody Core Facility, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany rb- α- CRY1 ARP59758_P050 Rabbit polyclonal hCRY1 sequence 151–200 IHC: 1:200 Aviva Systems Biology, San Diego, CA, USA Note: Identical amino acids to human CRY1 are shown in red.

Techniques: Immunofluorescence

Journal: The FASEB Journal

Article Title: Full‐Length Cryptochrome 1 in the Outer Segments of the Retinal Blue Cone Photoreceptors in Humans and Great Apes Suggests a Role Beyond Transcriptional Repression

doi: 10.1096/fj.202402614r

Figure Lengend Snippet: FIGURE 2 | Triple-labeling for full-length CRY1 (gp-α-CRY1 serum), for the SWS1 opsin of the blue cones, and for the LWS opsin of the green and red cones in central human retina (left column) and central bonobo retina (right column). Images from flatmounted retinal pieces, focused on the cone outer segments. In addition to the single-label images, merged images of the CRY1 label (in green) and the SWS1 opsin label (in magenta), and of the CRY1 label and the LWS opsin label (in red), are shown. The merged images confirm that the CRY1 label is limited to blue cones (whitish appearance) and does not occur in green and red cones (which would appear yellow). Photoreceptor outer segment preservation is less good in the human than in the bonobo tissue. The images are maximum intensity projections of confocal image stacks. Scale bar in the top left image is 50 μm and applies to all images.

Article Snippet: Antibody name Species and antibody clonality Immunization peptide Dilution Source gp- α- CRY1 Guinea pig polyclonal RPNPEEETQSVGPKVQRQST hCRY1 sequence 566–585 IHC: 1:100 WB: 1:500 Genovac GmbH, Freiburg, Germany [32] rb- α- CRY1 PA1- 527 Rabbit polyclonal QSVGPKVQRQSSN hCRY1 sequence 574–586 IHC: 1:500 WB: 1:1000 Thermo Fisher Scientific, Waltham, MA, USA rt- α- CRY1 3E12 Rat monoclonal RPNPEEETQSVGPKVQRQST hCRY1 sequence 566–585 IHC: undiluted Monoclonal Antibody Core Facility, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany [33] rt- α- CRY1 17A2 Rat monoclonal TTPVSDDHDEKYG hCRY1 sequence 179–191 IHC: 1:2 WB 1:1000 This study; Monoclonal Antibody Core Facility, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany rb- α- CRY1 ARP59758_P050 Rabbit polyclonal hCRY1 sequence 151–200 IHC: 1:200 Aviva Systems Biology, San Diego, CA, USA Note: Identical amino acids to human CRY1 are shown in red.

Techniques: Labeling, Preserving

Journal: The FASEB Journal

Article Title: Full‐Length Cryptochrome 1 in the Outer Segments of the Retinal Blue Cone Photoreceptors in Humans and Great Apes Suggests a Role Beyond Transcriptional Repression

doi: 10.1096/fj.202402614r

Figure Lengend Snippet: FIGURE 4 | Western Blot of GFP and CRY1-GFP expressed in HEK 293 cells. 40 μg of HEK cell lysate expressing either GFP alone or a CRY1- GFP fusion protein was subjected to SDS-PAGE and Western blotting. A Western blot incubated with the gp-α-CRY1 serum shows a band close to the expected size of 93 kDa, but not in HEK cells expressing GFP only (A). Other unspecific bands seem to come from the secondary antibody, as demonstrated in the Western blot incubated with the secondary antibody only (B). A Western blot incubated with an anti-TurboGFP antibody was used as a positive control (C). *This band likely indicates a truncated C-terminus with the GFP-tag. **The red color of the 70 kDa protein marker appears bright white with illumination.

Article Snippet: Antibody name Species and antibody clonality Immunization peptide Dilution Source gp- α- CRY1 Guinea pig polyclonal RPNPEEETQSVGPKVQRQST hCRY1 sequence 566–585 IHC: 1:100 WB: 1:500 Genovac GmbH, Freiburg, Germany [32] rb- α- CRY1 PA1- 527 Rabbit polyclonal QSVGPKVQRQSSN hCRY1 sequence 574–586 IHC: 1:500 WB: 1:1000 Thermo Fisher Scientific, Waltham, MA, USA rt- α- CRY1 3E12 Rat monoclonal RPNPEEETQSVGPKVQRQST hCRY1 sequence 566–585 IHC: undiluted Monoclonal Antibody Core Facility, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany [33] rt- α- CRY1 17A2 Rat monoclonal TTPVSDDHDEKYG hCRY1 sequence 179–191 IHC: 1:2 WB 1:1000 This study; Monoclonal Antibody Core Facility, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany rb- α- CRY1 ARP59758_P050 Rabbit polyclonal hCRY1 sequence 151–200 IHC: 1:200 Aviva Systems Biology, San Diego, CA, USA Note: Identical amino acids to human CRY1 are shown in red.

Techniques: Western Blot, Expressing, SDS Page, Incubation, Positive Control, Marker

Journal: The FASEB Journal

Article Title: Full‐Length Cryptochrome 1 in the Outer Segments of the Retinal Blue Cone Photoreceptors in Humans and Great Apes Suggests a Role Beyond Transcriptional Repression

doi: 10.1096/fj.202402614r

Figure Lengend Snippet: FIGURE 3 | Full-length CRY1 label (detected with rt-α-CRY1 3E12) in the SWS1 (blue) cones of bonobo and human retinae in transverse sections. Left column: CRY1 immunofluorescence (green). Middle column: SWS1 opsin immunofluorescence located in the blue cone outer segments, and in bonobo also more faintly across the whole blue cones (magenta). Right column: Merged images, showing that CRY1 and SWS1 opsin colocalize in the blue cone outer segments. The human tissue preservation is less good. The images are maximum intensity projections of confocal image stacks. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, IS, photoreceptor outer and inner segments. Scale bar in the top left image is 100 μm and applies to all images.

Article Snippet: Antibody name Species and antibody clonality Immunization peptide Dilution Source gp- α- CRY1 Guinea pig polyclonal RPNPEEETQSVGPKVQRQST hCRY1 sequence 566–585 IHC: 1:100 WB: 1:500 Genovac GmbH, Freiburg, Germany [32] rb- α- CRY1 PA1- 527 Rabbit polyclonal QSVGPKVQRQSSN hCRY1 sequence 574–586 IHC: 1:500 WB: 1:1000 Thermo Fisher Scientific, Waltham, MA, USA rt- α- CRY1 3E12 Rat monoclonal RPNPEEETQSVGPKVQRQST hCRY1 sequence 566–585 IHC: undiluted Monoclonal Antibody Core Facility, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany [33] rt- α- CRY1 17A2 Rat monoclonal TTPVSDDHDEKYG hCRY1 sequence 179–191 IHC: 1:2 WB 1:1000 This study; Monoclonal Antibody Core Facility, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany rb- α- CRY1 ARP59758_P050 Rabbit polyclonal hCRY1 sequence 151–200 IHC: 1:200 Aviva Systems Biology, San Diego, CA, USA Note: Identical amino acids to human CRY1 are shown in red.

Techniques: Immunofluorescence, Preserving

Journal: The FASEB Journal

Article Title: Full‐Length Cryptochrome 1 in the Outer Segments of the Retinal Blue Cone Photoreceptors in Humans and Great Apes Suggests a Role Beyond Transcriptional Repression

doi: 10.1096/fj.202402614r

Figure Lengend Snippet: FIGURE 5 | Western blot of hCRY1-GFP or hCRY1Δ566-585- CNGB141-50-GFP expressed in HEK 293 cells. 40 μg of HEK 293 cell lysate expressing the empty plasmid (GFP), a CRY1-GFP fusion pro- tein, or a hCRY1Δ566-585-CNGB141-50-GFP chimeric fusion protein were analyzed using SDS-PAGE and Western blotting. The gp-α-CRY1 se- rum used in this study recognized only full-length CRY1, but not the hCRY1Δ566-585-CNGB141-50-GFP chimeric protein. Interestingly, the monoclonal rt-α-CRY1 17A2 directed against an N-terminal region of CRY1 recognized the truncated CRY1 protein much better than the full- length protein. A control staining against TurboGFP indicates that both proteins were expressed in approximately equal amounts. Full Western blot images can be found in the supplements (Figure S3).

Article Snippet: Antibody name Species and antibody clonality Immunization peptide Dilution Source gp- α- CRY1 Guinea pig polyclonal RPNPEEETQSVGPKVQRQST hCRY1 sequence 566–585 IHC: 1:100 WB: 1:500 Genovac GmbH, Freiburg, Germany [32] rb- α- CRY1 PA1- 527 Rabbit polyclonal QSVGPKVQRQSSN hCRY1 sequence 574–586 IHC: 1:500 WB: 1:1000 Thermo Fisher Scientific, Waltham, MA, USA rt- α- CRY1 3E12 Rat monoclonal RPNPEEETQSVGPKVQRQST hCRY1 sequence 566–585 IHC: undiluted Monoclonal Antibody Core Facility, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany [33] rt- α- CRY1 17A2 Rat monoclonal TTPVSDDHDEKYG hCRY1 sequence 179–191 IHC: 1:2 WB 1:1000 This study; Monoclonal Antibody Core Facility, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany rb- α- CRY1 ARP59758_P050 Rabbit polyclonal hCRY1 sequence 151–200 IHC: 1:200 Aviva Systems Biology, San Diego, CA, USA Note: Identical amino acids to human CRY1 are shown in red.

Techniques: Western Blot, Expressing, Plasmid Preparation, SDS Page, Control, Staining

Journal: The FASEB Journal

Article Title: Full‐Length Cryptochrome 1 in the Outer Segments of the Retinal Blue Cone Photoreceptors in Humans and Great Apes Suggests a Role Beyond Transcriptional Repression

doi: 10.1096/fj.202402614r

Figure Lengend Snippet: FIGURE 6 | Subcellular fractionation of HEK cells with and without transfected hCRY1. Cell lysates of the cytosolic fraction (C), membrane fraction (M), and nuclear fraction (N) were subjected to SDS-PAGE and Western blotting and probed against GAPDH as a marker for cytoso- lic and membrane fractions, against Na/K-ATPase as a marker for the membrane fraction, and against Histone H3 as a nuclear marker. The CRY1 antiserum used in this Western blot was rb-α-CRY1 PA1-527, rec- ognizing full-length CRY1. Full Western blot images can be found in the supplements (Figure S4).

Article Snippet: Antibody name Species and antibody clonality Immunization peptide Dilution Source gp- α- CRY1 Guinea pig polyclonal RPNPEEETQSVGPKVQRQST hCRY1 sequence 566–585 IHC: 1:100 WB: 1:500 Genovac GmbH, Freiburg, Germany [32] rb- α- CRY1 PA1- 527 Rabbit polyclonal QSVGPKVQRQSSN hCRY1 sequence 574–586 IHC: 1:500 WB: 1:1000 Thermo Fisher Scientific, Waltham, MA, USA rt- α- CRY1 3E12 Rat monoclonal RPNPEEETQSVGPKVQRQST hCRY1 sequence 566–585 IHC: undiluted Monoclonal Antibody Core Facility, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany [33] rt- α- CRY1 17A2 Rat monoclonal TTPVSDDHDEKYG hCRY1 sequence 179–191 IHC: 1:2 WB 1:1000 This study; Monoclonal Antibody Core Facility, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany rb- α- CRY1 ARP59758_P050 Rabbit polyclonal hCRY1 sequence 151–200 IHC: 1:200 Aviva Systems Biology, San Diego, CA, USA Note: Identical amino acids to human CRY1 are shown in red.

Techniques: Fractionation, Transfection, Membrane, SDS Page, Western Blot, Marker