Western Blot:Article Title: Keratin isoform shifts modulate motility signals during wound healing
Article Snippet: .. Antibody Application and dilution Mouse anti-keratin 1 (Novus LHK1) WB, 1:250 Rabbit anti-keratin 5 (Abcam ab52635), N-terminal epitope IF/PLA, 1:200 WB, 1:10,000 IHC, 1:200 Rabbit anti-keratin 5 (Cell Signaling Technology #25807), C-terminal epitope WB, 1:1,000 Rabbit anti-keratin 6 (Abcam ab93279), epitope conserved in K6A, K6B, and K6C IF, 1:50 WB, 1:2,000 IHC, 1:50 Rabbit anti-keratin 10 (Abcam ab76318) WB, 1:1,000 Mouse anti-keratin 14 (EMD Millipore MAB3232) WB, 1:200 Mouse anti-keratin 16 (Invitrogen LL025) WB, 1:500 Rabbit polyclonal anti-keratin 17 (Abcam ab53707) IF/PLA, 1:1,000 WB, 1:1,000 IHC, 1:100 Mouse anti-beta-actin (Sigma A1978) WB, 1:5,000 Rabbit polyclonal anti-non-muscle myosin heavy chain IIA (MYH9; Invitrogen PA5-17025) PLA, 1:200 Mouse anti-phospho-myosin regulatory light chain (Cell Signaling Technology #3675) PLA, 1:100 Mouse anti-vimentin (Sigma V6630) IF/PLA, 1:100 Rabbit anti-vimentin (Cell Signaling Technology #5741) WB, 1:1,000 Rabbit anti-ROCK2 and ROCK1 (Abcam ab45171) PLA, 1:250 Rabbit polyclonal anti-myosin light chain kinase (Invitrogen PA5-76982) PLA, 1:200 HRP, goat anti- mouse IgG (Invitrogan #31430) WB, 1:1,000 HRP, goat anti- rabbit IgG (Invitrogan #31460) WB, 1:1,000 Alexa Fluor 488, goat anti- mouse IgG (Invitrogen A-11209) IF, 1:500 Alexa Fluor 488, goat anti- rabbit IgG (Invitrogen A-11008) IF, 1:500 Alexa Fluor 568, goat anti- mouse IgG (Invitrogen A-11004) IF, 1:500 Alexa Fluor 568, goat anti- rabbit IgG (Invitrogen A-11036) IF, 1:500 Alexa Fluor 647, goat anti- rabbit IgG (Invitrogen A-21244) IF, 1:500 IF, immunofluorescence; IHC, immunohistochemistry or immunofluorescence on formalin-fixed paraffin-embedded samples; PLA, proximity ligation assay; WB, western blot Nanes et al. | Keratin isoforms modulate motility signals | 23 B 50 K5 Basal Differentiated Wound 50 50 50 50 50 50 14 K1 K10 K6 K16 K17 K5 hig h K6 A hig h K5 hig h K6 A hig h 50kDK16 50kDK17 37kD β-actin K5 hig h K6 A hig h K6 A hig h K5 hig h 50kDK14 37kDβ-actin K5 K5mNG K6A K6AmRb K14 K1 K10 K6B K6C K16 K17 W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h 10−2 10−1 100 101 102 Cell Line Fo ld e xp re ss io n 50kD 37kD β-actin K5 K5-G K6 K6-R 75kD 50kD 75kD 100kD K5 hig h K5 hig h K5 hig h K6 A hig h K6 A hig h K6 A hig h Figure S1. ..
Immunohistochemistry:Article Title: Keratin isoform shifts modulate motility signals during wound healing
Article Snippet: .. Antibody Application and dilution Mouse anti-keratin 1 (Novus LHK1) WB, 1:250 Rabbit anti-keratin 5 (Abcam ab52635), N-terminal epitope IF/PLA, 1:200 WB, 1:10,000 IHC, 1:200 Rabbit anti-keratin 5 (Cell Signaling Technology #25807), C-terminal epitope WB, 1:1,000 Rabbit anti-keratin 6 (Abcam ab93279), epitope conserved in K6A, K6B, and K6C IF, 1:50 WB, 1:2,000 IHC, 1:50 Rabbit anti-keratin 10 (Abcam ab76318) WB, 1:1,000 Mouse anti-keratin 14 (EMD Millipore MAB3232) WB, 1:200 Mouse anti-keratin 16 (Invitrogen LL025) WB, 1:500 Rabbit polyclonal anti-keratin 17 (Abcam ab53707) IF/PLA, 1:1,000 WB, 1:1,000 IHC, 1:100 Mouse anti-beta-actin (Sigma A1978) WB, 1:5,000 Rabbit polyclonal anti-non-muscle myosin heavy chain IIA (MYH9; Invitrogen PA5-17025) PLA, 1:200 Mouse anti-phospho-myosin regulatory light chain (Cell Signaling Technology #3675) PLA, 1:100 Mouse anti-vimentin (Sigma V6630) IF/PLA, 1:100 Rabbit anti-vimentin (Cell Signaling Technology #5741) WB, 1:1,000 Rabbit anti-ROCK2 and ROCK1 (Abcam ab45171) PLA, 1:250 Rabbit polyclonal anti-myosin light chain kinase (Invitrogen PA5-76982) PLA, 1:200 HRP, goat anti- mouse IgG (Invitrogan #31430) WB, 1:1,000 HRP, goat anti- rabbit IgG (Invitrogan #31460) WB, 1:1,000 Alexa Fluor 488, goat anti- mouse IgG (Invitrogen A-11209) IF, 1:500 Alexa Fluor 488, goat anti- rabbit IgG (Invitrogen A-11008) IF, 1:500 Alexa Fluor 568, goat anti- mouse IgG (Invitrogen A-11004) IF, 1:500 Alexa Fluor 568, goat anti- rabbit IgG (Invitrogen A-11036) IF, 1:500 Alexa Fluor 647, goat anti- rabbit IgG (Invitrogen A-21244) IF, 1:500 IF, immunofluorescence; IHC, immunohistochemistry or immunofluorescence on formalin-fixed paraffin-embedded samples; PLA, proximity ligation assay; WB, western blot Nanes et al. | Keratin isoforms modulate motility signals | 23 B 50 K5 Basal Differentiated Wound 50 50 50 50 50 50 14 K1 K10 K6 K16 K17 K5 hig h K6 A hig h K5 hig h K6 A hig h 50kDK16 50kDK17 37kD β-actin K5 hig h K6 A hig h K6 A hig h K5 hig h 50kDK14 37kDβ-actin K5 K5mNG K6A K6AmRb K14 K1 K10 K6B K6C K16 K17 W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h 10−2 10−1 100 101 102 Cell Line Fo ld e xp re ss io n 50kD 37kD β-actin K5 K5-G K6 K6-R 75kD 50kD 75kD 100kD K5 hig h K5 hig h K5 hig h K6 A hig h K6 A hig h K6 A hig h Figure S1. ..
Proximity Ligation Assay:Article Title: Keratin isoform shifts modulate motility signals during wound healing
Article Snippet: .. Antibody Application and dilution Mouse anti-keratin 1 (Novus LHK1) WB, 1:250 Rabbit anti-keratin 5 (Abcam ab52635), N-terminal epitope IF/PLA, 1:200 WB, 1:10,000 IHC, 1:200 Rabbit anti-keratin 5 (Cell Signaling Technology #25807), C-terminal epitope WB, 1:1,000 Rabbit anti-keratin 6 (Abcam ab93279), epitope conserved in K6A, K6B, and K6C IF, 1:50 WB, 1:2,000 IHC, 1:50 Rabbit anti-keratin 10 (Abcam ab76318) WB, 1:1,000 Mouse anti-keratin 14 (EMD Millipore MAB3232) WB, 1:200 Mouse anti-keratin 16 (Invitrogen LL025) WB, 1:500 Rabbit polyclonal anti-keratin 17 (Abcam ab53707) IF/PLA, 1:1,000 WB, 1:1,000 IHC, 1:100 Mouse anti-beta-actin (Sigma A1978) WB, 1:5,000 Rabbit polyclonal anti-non-muscle myosin heavy chain IIA (MYH9; Invitrogen PA5-17025) PLA, 1:200 Mouse anti-phospho-myosin regulatory light chain (Cell Signaling Technology #3675) PLA, 1:100 Mouse anti-vimentin (Sigma V6630) IF/PLA, 1:100 Rabbit anti-vimentin (Cell Signaling Technology #5741) WB, 1:1,000 Rabbit anti-ROCK2 and ROCK1 (Abcam ab45171) PLA, 1:250 Rabbit polyclonal anti-myosin light chain kinase (Invitrogen PA5-76982) PLA, 1:200 HRP, goat anti- mouse IgG (Invitrogan #31430) WB, 1:1,000 HRP, goat anti- rabbit IgG (Invitrogan #31460) WB, 1:1,000 Alexa Fluor 488, goat anti- mouse IgG (Invitrogen A-11209) IF, 1:500 Alexa Fluor 488, goat anti- rabbit IgG (Invitrogen A-11008) IF, 1:500 Alexa Fluor 568, goat anti- mouse IgG (Invitrogen A-11004) IF, 1:500 Alexa Fluor 568, goat anti- rabbit IgG (Invitrogen A-11036) IF, 1:500 Alexa Fluor 647, goat anti- rabbit IgG (Invitrogen A-21244) IF, 1:500 IF, immunofluorescence; IHC, immunohistochemistry or immunofluorescence on formalin-fixed paraffin-embedded samples; PLA, proximity ligation assay; WB, western blot Nanes et al. | Keratin isoforms modulate motility signals | 23 B 50 K5 Basal Differentiated Wound 50 50 50 50 50 50 14 K1 K10 K6 K16 K17 K5 hig h K6 A hig h K5 hig h K6 A hig h 50kDK16 50kDK17 37kD β-actin K5 hig h K6 A hig h K6 A hig h K5 hig h 50kDK14 37kDβ-actin K5 K5mNG K6A K6AmRb K14 K1 K10 K6B K6C K16 K17 W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h 10−2 10−1 100 101 102 Cell Line Fo ld e xp re ss io n 50kD 37kD β-actin K5 K5-G K6 K6-R 75kD 50kD 75kD 100kD K5 hig h K5 hig h K5 hig h K6 A hig h K6 A hig h K6 A hig h Figure S1. ..
Immunofluorescence:Article Title: Keratin isoform shifts modulate motility signals during wound healing
Article Snippet: .. Antibody Application and dilution Mouse anti-keratin 1 (Novus LHK1) WB, 1:250 Rabbit anti-keratin 5 (Abcam ab52635), N-terminal epitope IF/PLA, 1:200 WB, 1:10,000 IHC, 1:200 Rabbit anti-keratin 5 (Cell Signaling Technology #25807), C-terminal epitope WB, 1:1,000 Rabbit anti-keratin 6 (Abcam ab93279), epitope conserved in K6A, K6B, and K6C IF, 1:50 WB, 1:2,000 IHC, 1:50 Rabbit anti-keratin 10 (Abcam ab76318) WB, 1:1,000 Mouse anti-keratin 14 (EMD Millipore MAB3232) WB, 1:200 Mouse anti-keratin 16 (Invitrogen LL025) WB, 1:500 Rabbit polyclonal anti-keratin 17 (Abcam ab53707) IF/PLA, 1:1,000 WB, 1:1,000 IHC, 1:100 Mouse anti-beta-actin (Sigma A1978) WB, 1:5,000 Rabbit polyclonal anti-non-muscle myosin heavy chain IIA (MYH9; Invitrogen PA5-17025) PLA, 1:200 Mouse anti-phospho-myosin regulatory light chain (Cell Signaling Technology #3675) PLA, 1:100 Mouse anti-vimentin (Sigma V6630) IF/PLA, 1:100 Rabbit anti-vimentin (Cell Signaling Technology #5741) WB, 1:1,000 Rabbit anti-ROCK2 and ROCK1 (Abcam ab45171) PLA, 1:250 Rabbit polyclonal anti-myosin light chain kinase (Invitrogen PA5-76982) PLA, 1:200 HRP, goat anti- mouse IgG (Invitrogan #31430) WB, 1:1,000 HRP, goat anti- rabbit IgG (Invitrogan #31460) WB, 1:1,000 Alexa Fluor 488, goat anti- mouse IgG (Invitrogen A-11209) IF, 1:500 Alexa Fluor 488, goat anti- rabbit IgG (Invitrogen A-11008) IF, 1:500 Alexa Fluor 568, goat anti- mouse IgG (Invitrogen A-11004) IF, 1:500 Alexa Fluor 568, goat anti- rabbit IgG (Invitrogen A-11036) IF, 1:500 Alexa Fluor 647, goat anti- rabbit IgG (Invitrogen A-21244) IF, 1:500 IF, immunofluorescence; IHC, immunohistochemistry or immunofluorescence on formalin-fixed paraffin-embedded samples; PLA, proximity ligation assay; WB, western blot Nanes et al. | Keratin isoforms modulate motility signals | 23 B 50 K5 Basal Differentiated Wound 50 50 50 50 50 50 14 K1 K10 K6 K16 K17 K5 hig h K6 A hig h K5 hig h K6 A hig h 50kDK16 50kDK17 37kD β-actin K5 hig h K6 A hig h K6 A hig h K5 hig h 50kDK14 37kDβ-actin K5 K5mNG K6A K6AmRb K14 K1 K10 K6B K6C K16 K17 W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h 10−2 10−1 100 101 102 Cell Line Fo ld e xp re ss io n 50kD 37kD β-actin K5 K5-G K6 K6-R 75kD 50kD 75kD 100kD K5 hig h K5 hig h K5 hig h K6 A hig h K6 A hig h K6 A hig h Figure S1. ..
Formalin-fixed Paraffin-Embedded:Article Title: Keratin isoform shifts modulate motility signals during wound healing
Article Snippet: .. Antibody Application and dilution Mouse anti-keratin 1 (Novus LHK1) WB, 1:250 Rabbit anti-keratin 5 (Abcam ab52635), N-terminal epitope IF/PLA, 1:200 WB, 1:10,000 IHC, 1:200 Rabbit anti-keratin 5 (Cell Signaling Technology #25807), C-terminal epitope WB, 1:1,000 Rabbit anti-keratin 6 (Abcam ab93279), epitope conserved in K6A, K6B, and K6C IF, 1:50 WB, 1:2,000 IHC, 1:50 Rabbit anti-keratin 10 (Abcam ab76318) WB, 1:1,000 Mouse anti-keratin 14 (EMD Millipore MAB3232) WB, 1:200 Mouse anti-keratin 16 (Invitrogen LL025) WB, 1:500 Rabbit polyclonal anti-keratin 17 (Abcam ab53707) IF/PLA, 1:1,000 WB, 1:1,000 IHC, 1:100 Mouse anti-beta-actin (Sigma A1978) WB, 1:5,000 Rabbit polyclonal anti-non-muscle myosin heavy chain IIA (MYH9; Invitrogen PA5-17025) PLA, 1:200 Mouse anti-phospho-myosin regulatory light chain (Cell Signaling Technology #3675) PLA, 1:100 Mouse anti-vimentin (Sigma V6630) IF/PLA, 1:100 Rabbit anti-vimentin (Cell Signaling Technology #5741) WB, 1:1,000 Rabbit anti-ROCK2 and ROCK1 (Abcam ab45171) PLA, 1:250 Rabbit polyclonal anti-myosin light chain kinase (Invitrogen PA5-76982) PLA, 1:200 HRP, goat anti- mouse IgG (Invitrogan #31430) WB, 1:1,000 HRP, goat anti- rabbit IgG (Invitrogan #31460) WB, 1:1,000 Alexa Fluor 488, goat anti- mouse IgG (Invitrogen A-11209) IF, 1:500 Alexa Fluor 488, goat anti- rabbit IgG (Invitrogen A-11008) IF, 1:500 Alexa Fluor 568, goat anti- mouse IgG (Invitrogen A-11004) IF, 1:500 Alexa Fluor 568, goat anti- rabbit IgG (Invitrogen A-11036) IF, 1:500 Alexa Fluor 647, goat anti- rabbit IgG (Invitrogen A-21244) IF, 1:500 IF, immunofluorescence; IHC, immunohistochemistry or immunofluorescence on formalin-fixed paraffin-embedded samples; PLA, proximity ligation assay; WB, western blot Nanes et al. | Keratin isoforms modulate motility signals | 23 B 50 K5 Basal Differentiated Wound 50 50 50 50 50 50 14 K1 K10 K6 K16 K17 K5 hig h K6 A hig h K5 hig h K6 A hig h 50kDK16 50kDK17 37kD β-actin K5 hig h K6 A hig h K6 A hig h K5 hig h 50kDK14 37kDβ-actin K5 K5mNG K6A K6AmRb K14 K1 K10 K6B K6C K16 K17 W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h W T K5 hi gh K6 Ah ig h 10−2 10−1 100 101 102 Cell Line Fo ld e xp re ss io n 50kD 37kD β-actin K5 K5-G K6 K6-R 75kD 50kD 75kD 100kD K5 hig h K5 hig h K5 hig h K6 A hig h K6 A hig h K6 A hig h Figure S1. ..
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