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Primary antibodies used in this study
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(A) Schwann cells isolated from P2 and 25-mo rats were treated with amino acid and serum-deficient starvation (Stv) medium, or subjected to HS and allowed to recover at 37 °C for 6 h. Steady-state expression of HSP90, HSP70 and <t>HSP40</t> were analyzed in whole cell lysates (15 µg/lane) of untreated control (Ct), Stv- or HS-treated samples. Blots were reprobed with anti-GAPDH antibody as a protein loading control. Molecular mass at the left, in kDa. (B) Schwann cells isolated from P2 and 25-mo old rats were subjected to HS (45 °C; 20 min) and allowed to recover at 37 °C for 6 h (HS+6h). Control untreated cells (Ct) and HS+6h samples were immunolabeled with anti-HSP70 (green) antibody. Hoechst dye (blue) was used to visualize nuclei. Scale bar, 10 µm.
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Primary antibodies used in this study

Journal:

Article Title: The molecular architecture of myelinated peripheral nerves is supported by calorie restriction with aging

doi: 10.1111/j.1474-9726.2009.00460.x

Figure Lengend Snippet: Primary antibodies used in this study

Article Snippet: To determine statistical significance between samples, unpaired t-test was performed using GraphPad Prism v5.0 software.Graph pad Prism software. table ft1 table-wrap mode="anchored" t5 caption a7 Species Antigen Source and Catalog # Dilution WB IS Chicken P0 Gift from Dr.Gerry Shaw, UF 1:10000 1:500 Rabbit PMP22 Pareek et. al, 1997; n/a 1:1000 n/a Rat MBP Chemicon, Temecula; MAB386 1:5000 n/a Mouse GAPDH Encor Biotechnology Inc.; MCA-1D4 1:5000 n/a Mouse NF-H Encor Biotechnology Inc.; MCA-NAP4 1:500 n/a Chicken NF-M Encor Biotechnology Inc.; CPCA-NF-M 1:5000 n/a Chicken NF-L Encor Biotechnology Inc.; CPCA-NF-L 1:5000 1:1000 Mouse Vimentin Sigma, St Louis, MO, USA; clone VIM 13.2 1:1000 1:500 Chicken MBP Encor Biotechnology, Inc.; CPCA-MBP n/a 1:500 Rabbit p75 NTR Chemicon; AB1554 1:10000 n/a Mouse pHH3 (Ser10) Millipore, Temecula; 05–598 1:500 1:200 Rabbit Pan-Na v (α-subunit) Chemicon; AB5210 1:250 1:100 Rabbit Kv1.1 Chemicon; AB5174 1:250 1:100 Rabbit HSF1 Stressgen; Victoria British Columbia; SPA- 901 1:500 1:250 Rat HSP90 Stressgen, SPA-835 1:2000 n/a Rabbit HSP70 Stressgen; SPA-812 1:1000 1:500 Rabbit HSP40 Stressgen; SPA-400 1:2000 n/a Goat HSP27 Santa Cruz Biotechnology, Inc.; sc-1049 1:2000 n/a Rabbit αB-crystallin Stressgen; SPA-223 1:1000 n/a Rabbit LAMP1 Gift from Dr. William Dunn Jr, UF 1:2000 n/a Rabbit Atg7 Gift from Dr. William Dunn Jr, UF 1:3000 n/a Rabbit pS6 (Ser235/236) Cell Signaling Technology, Inc., 2211 1:800 n/a Mouse S6 Cell Signaling Technology, Inc.; 2317 1:800 n/a Mouse HSP70 Stressgen; SPA-810 1:1000 1:250 Mouse LAMP1 Stressgen; VAM-EN001 n/a 1:250 Rabbit Cathepsin-D Cortex Biochem, San Leanardo, CA, USA; CP3090 1:1000 n/a Rabbit LC3 Cell Signaling Technology; 2775 1:1000 1:200 Open in a separate window WB: Western Blot; IS: Immunostaining; n/a: non-applicable Primary antibodies used in this study

Techniques:

(A) Schwann cells isolated from P2 and 25-mo rats were treated with amino acid and serum-deficient starvation (Stv) medium, or subjected to HS and allowed to recover at 37 °C for 6 h. Steady-state expression of HSP90, HSP70 and HSP40 were analyzed in whole cell lysates (15 µg/lane) of untreated control (Ct), Stv- or HS-treated samples. Blots were reprobed with anti-GAPDH antibody as a protein loading control. Molecular mass at the left, in kDa. (B) Schwann cells isolated from P2 and 25-mo old rats were subjected to HS (45 °C; 20 min) and allowed to recover at 37 °C for 6 h (HS+6h). Control untreated cells (Ct) and HS+6h samples were immunolabeled with anti-HSP70 (green) antibody. Hoechst dye (blue) was used to visualize nuclei. Scale bar, 10 µm.

Journal:

Article Title: The molecular architecture of myelinated peripheral nerves is supported by calorie restriction with aging

doi: 10.1111/j.1474-9726.2009.00460.x

Figure Lengend Snippet: (A) Schwann cells isolated from P2 and 25-mo rats were treated with amino acid and serum-deficient starvation (Stv) medium, or subjected to HS and allowed to recover at 37 °C for 6 h. Steady-state expression of HSP90, HSP70 and HSP40 were analyzed in whole cell lysates (15 µg/lane) of untreated control (Ct), Stv- or HS-treated samples. Blots were reprobed with anti-GAPDH antibody as a protein loading control. Molecular mass at the left, in kDa. (B) Schwann cells isolated from P2 and 25-mo old rats were subjected to HS (45 °C; 20 min) and allowed to recover at 37 °C for 6 h (HS+6h). Control untreated cells (Ct) and HS+6h samples were immunolabeled with anti-HSP70 (green) antibody. Hoechst dye (blue) was used to visualize nuclei. Scale bar, 10 µm.

Article Snippet: To determine statistical significance between samples, unpaired t-test was performed using GraphPad Prism v5.0 software.Graph pad Prism software. table ft1 table-wrap mode="anchored" t5 caption a7 Species Antigen Source and Catalog # Dilution WB IS Chicken P0 Gift from Dr.Gerry Shaw, UF 1:10000 1:500 Rabbit PMP22 Pareek et. al, 1997; n/a 1:1000 n/a Rat MBP Chemicon, Temecula; MAB386 1:5000 n/a Mouse GAPDH Encor Biotechnology Inc.; MCA-1D4 1:5000 n/a Mouse NF-H Encor Biotechnology Inc.; MCA-NAP4 1:500 n/a Chicken NF-M Encor Biotechnology Inc.; CPCA-NF-M 1:5000 n/a Chicken NF-L Encor Biotechnology Inc.; CPCA-NF-L 1:5000 1:1000 Mouse Vimentin Sigma, St Louis, MO, USA; clone VIM 13.2 1:1000 1:500 Chicken MBP Encor Biotechnology, Inc.; CPCA-MBP n/a 1:500 Rabbit p75 NTR Chemicon; AB1554 1:10000 n/a Mouse pHH3 (Ser10) Millipore, Temecula; 05–598 1:500 1:200 Rabbit Pan-Na v (α-subunit) Chemicon; AB5210 1:250 1:100 Rabbit Kv1.1 Chemicon; AB5174 1:250 1:100 Rabbit HSF1 Stressgen; Victoria British Columbia; SPA- 901 1:500 1:250 Rat HSP90 Stressgen, SPA-835 1:2000 n/a Rabbit HSP70 Stressgen; SPA-812 1:1000 1:500 Rabbit HSP40 Stressgen; SPA-400 1:2000 n/a Goat HSP27 Santa Cruz Biotechnology, Inc.; sc-1049 1:2000 n/a Rabbit αB-crystallin Stressgen; SPA-223 1:1000 n/a Rabbit LAMP1 Gift from Dr. William Dunn Jr, UF 1:2000 n/a Rabbit Atg7 Gift from Dr. William Dunn Jr, UF 1:3000 n/a Rabbit pS6 (Ser235/236) Cell Signaling Technology, Inc., 2211 1:800 n/a Mouse S6 Cell Signaling Technology, Inc.; 2317 1:800 n/a Mouse HSP70 Stressgen; SPA-810 1:1000 1:250 Mouse LAMP1 Stressgen; VAM-EN001 n/a 1:250 Rabbit Cathepsin-D Cortex Biochem, San Leanardo, CA, USA; CP3090 1:1000 n/a Rabbit LC3 Cell Signaling Technology; 2775 1:1000 1:200 Open in a separate window WB: Western Blot; IS: Immunostaining; n/a: non-applicable Primary antibodies used in this study

Techniques: Isolation, Expressing, Control, Immunolabeling

(A) Total sciatic nerve lysates (25 µg/lane) from the indicated ages and diet were analyzed with anti-heat shock factor 1 (HSF1) antibody. The same nerve lysates (20 µg/lane) were also probed with antibodies against HSP90, HSP70, HSP40, HSP27 and αB-crystallin. (B) Quantification of HSF1 and HSP90 protein levels normalized to GAPDH from three independent experiments (##p<0.01, ###p<0.001, Fisher’s PLSD, mean ± SEM), AU: arbitrary units. The effect of CR on these proteins was analyzed by comparing HSF1 or HSP90 protein values with age-matched AL counterparts (*p<0.05, **p<0.05, unpaired t-test, mean ± SEM). (C) Steady-state levels of LAMP1, Atg7, pS6 and S6 proteins in sciatic nerves from AL and CR rats were analyzed by Western blots. Blots were reprobed with anti-GAPDH antibody as protein loading control. Molecular mass at the left, in kDa. (D) Quantification of LAMP1 and Atg7 protein levels normalized to GAPDH from three independent experiments (#p<0.05, ###p<0.001, Fisher’s PLSD analysis, *p<0.05, unpaired t-test, mean ± SEM). (E) Blots of pS6 and S6 from three independent experiments were quantified and the values are represented as ratio of pS6/S6. The pS6/S6 ratio of 8-mo old AL sample was set as 1 (*p<0.05, unpaired t-test, mean ± SEM). A-D, n=3 rats per condition.

Journal:

Article Title: The molecular architecture of myelinated peripheral nerves is supported by calorie restriction with aging

doi: 10.1111/j.1474-9726.2009.00460.x

Figure Lengend Snippet: (A) Total sciatic nerve lysates (25 µg/lane) from the indicated ages and diet were analyzed with anti-heat shock factor 1 (HSF1) antibody. The same nerve lysates (20 µg/lane) were also probed with antibodies against HSP90, HSP70, HSP40, HSP27 and αB-crystallin. (B) Quantification of HSF1 and HSP90 protein levels normalized to GAPDH from three independent experiments (##p<0.01, ###p<0.001, Fisher’s PLSD, mean ± SEM), AU: arbitrary units. The effect of CR on these proteins was analyzed by comparing HSF1 or HSP90 protein values with age-matched AL counterparts (*p<0.05, **p<0.05, unpaired t-test, mean ± SEM). (C) Steady-state levels of LAMP1, Atg7, pS6 and S6 proteins in sciatic nerves from AL and CR rats were analyzed by Western blots. Blots were reprobed with anti-GAPDH antibody as protein loading control. Molecular mass at the left, in kDa. (D) Quantification of LAMP1 and Atg7 protein levels normalized to GAPDH from three independent experiments (#p<0.05, ###p<0.001, Fisher’s PLSD analysis, *p<0.05, unpaired t-test, mean ± SEM). (E) Blots of pS6 and S6 from three independent experiments were quantified and the values are represented as ratio of pS6/S6. The pS6/S6 ratio of 8-mo old AL sample was set as 1 (*p<0.05, unpaired t-test, mean ± SEM). A-D, n=3 rats per condition.

Article Snippet: To determine statistical significance between samples, unpaired t-test was performed using GraphPad Prism v5.0 software.Graph pad Prism software. table ft1 table-wrap mode="anchored" t5 caption a7 Species Antigen Source and Catalog # Dilution WB IS Chicken P0 Gift from Dr.Gerry Shaw, UF 1:10000 1:500 Rabbit PMP22 Pareek et. al, 1997; n/a 1:1000 n/a Rat MBP Chemicon, Temecula; MAB386 1:5000 n/a Mouse GAPDH Encor Biotechnology Inc.; MCA-1D4 1:5000 n/a Mouse NF-H Encor Biotechnology Inc.; MCA-NAP4 1:500 n/a Chicken NF-M Encor Biotechnology Inc.; CPCA-NF-M 1:5000 n/a Chicken NF-L Encor Biotechnology Inc.; CPCA-NF-L 1:5000 1:1000 Mouse Vimentin Sigma, St Louis, MO, USA; clone VIM 13.2 1:1000 1:500 Chicken MBP Encor Biotechnology, Inc.; CPCA-MBP n/a 1:500 Rabbit p75 NTR Chemicon; AB1554 1:10000 n/a Mouse pHH3 (Ser10) Millipore, Temecula; 05–598 1:500 1:200 Rabbit Pan-Na v (α-subunit) Chemicon; AB5210 1:250 1:100 Rabbit Kv1.1 Chemicon; AB5174 1:250 1:100 Rabbit HSF1 Stressgen; Victoria British Columbia; SPA- 901 1:500 1:250 Rat HSP90 Stressgen, SPA-835 1:2000 n/a Rabbit HSP70 Stressgen; SPA-812 1:1000 1:500 Rabbit HSP40 Stressgen; SPA-400 1:2000 n/a Goat HSP27 Santa Cruz Biotechnology, Inc.; sc-1049 1:2000 n/a Rabbit αB-crystallin Stressgen; SPA-223 1:1000 n/a Rabbit LAMP1 Gift from Dr. William Dunn Jr, UF 1:2000 n/a Rabbit Atg7 Gift from Dr. William Dunn Jr, UF 1:3000 n/a Rabbit pS6 (Ser235/236) Cell Signaling Technology, Inc., 2211 1:800 n/a Mouse S6 Cell Signaling Technology, Inc.; 2317 1:800 n/a Mouse HSP70 Stressgen; SPA-810 1:1000 1:250 Mouse LAMP1 Stressgen; VAM-EN001 n/a 1:250 Rabbit Cathepsin-D Cortex Biochem, San Leanardo, CA, USA; CP3090 1:1000 n/a Rabbit LC3 Cell Signaling Technology; 2775 1:1000 1:200 Open in a separate window WB: Western Blot; IS: Immunostaining; n/a: non-applicable Primary antibodies used in this study

Techniques: Western Blot, Control

Primary antibodies used in this study

Journal:

Article Title: The molecular architecture of myelinated peripheral nerves is supported by calorie restriction with aging

doi: 10.1111/j.1474-9726.2009.00460.x

Figure Lengend Snippet: Primary antibodies used in this study

Article Snippet: To determine statistical significance between samples, unpaired t-test was performed using GraphPad Prism v5.0 software.Graph pad Prism software. table ft1 table-wrap mode="anchored" t5 caption a7 Species Antigen Source and Catalog # Dilution WB IS Chicken P0 Gift from Dr.Gerry Shaw, UF 1:10000 1:500 Rabbit PMP22 Pareek et. al, 1997; n/a 1:1000 n/a Rat MBP Chemicon, Temecula; MAB386 1:5000 n/a Mouse GAPDH Encor Biotechnology Inc.; MCA-1D4 1:5000 n/a Mouse NF-H Encor Biotechnology Inc.; MCA-NAP4 1:500 n/a Chicken NF-M Encor Biotechnology Inc.; CPCA-NF-M 1:5000 n/a Chicken NF-L Encor Biotechnology Inc.; CPCA-NF-L 1:5000 1:1000 Mouse Vimentin Sigma, St Louis, MO, USA; clone VIM 13.2 1:1000 1:500 Chicken MBP Encor Biotechnology, Inc.; CPCA-MBP n/a 1:500 Rabbit p75 NTR Chemicon; AB1554 1:10000 n/a Mouse pHH3 (Ser10) Millipore, Temecula; 05–598 1:500 1:200 Rabbit Pan-Na v (α-subunit) Chemicon; AB5210 1:250 1:100 Rabbit Kv1.1 Chemicon; AB5174 1:250 1:100 Rabbit HSF1 Stressgen; Victoria British Columbia; SPA- 901 1:500 1:250 Rat HSP90 Stressgen, SPA-835 1:2000 n/a Rabbit HSP70 Stressgen; SPA-812 1:1000 1:500 Rabbit HSP40 Stressgen; SPA-400 1:2000 n/a Goat HSP27 Santa Cruz Biotechnology, Inc.; sc-1049 1:2000 n/a Rabbit αB-crystallin Stressgen; SPA-223 1:1000 n/a Rabbit LAMP1 Gift from Dr. William Dunn Jr, UF 1:2000 n/a Rabbit Atg7 Gift from Dr. William Dunn Jr, UF 1:3000 n/a Rabbit pS6 (Ser235/236) Cell Signaling Technology, Inc., 2211 1:800 n/a Mouse S6 Cell Signaling Technology, Inc.; 2317 1:800 n/a Mouse HSP70 Stressgen; SPA-810 1:1000 1:250 Mouse LAMP1 Stressgen; VAM-EN001 n/a 1:250 Rabbit Cathepsin-D Cortex Biochem, San Leanardo, CA, USA; CP3090 1:1000 n/a Rabbit LC3 Cell Signaling Technology; 2775 1:1000 1:200 Open in a separate window WB: Western Blot; IS: Immunostaining; n/a: non-applicable Primary antibodies used in this study

Techniques: