Perature. Membranes were incubated overnight on a horizontal shaker at 4uC, with get Thiazole Orange primary antibodies diluted in blocking buffer. Primary antibodies used were collagen type 8 a2 (1:100), N-cadherin (1:200), p53 (1:200), and p16INK4 (1:100) (all Santa Cruz Biotechnology; Santa Cruz, CA), cyclin D (1:500; Millipore), CDK4 (1:200; Thermo ScientificH), Phospho-p53 (1:5000; Cell Signaling Technology; Danvers, MA) and mouse monoclonal b-actin (1:10000, Sigma Aldrich). Blots were rinsed with TBST 3 times for 10 min each and exposed to HRPconjugated donkey anti-mouse or -rabbit IgG for 1 hr at room temperature. All secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA) and diluted 1:2000 in blocking buffer. After washing 3 times in TBST, proteins were detected with SuperSignalH Pico or Femto chemiluminescent substrate (Thermo Scientific). A minimum of 2 different passages were analyzed per group and cell type.Telomeric Repeat Amplification Protocol (TRAP)TRAP assays were performed using the TRAPeze RT kit (Millipore) according to the manufacture’s protocol. Briefly, cells were harvested, washed with PBS, and stored at 280uC until use. After thawing, cells were immediately extracted with CHAPS lysis buffer (Millipore) and total protein concentration was determined using the BCATM Reducing Agent Compatible Assay (Pierce; Rockfort, IL) TRAP reactions were performed with 1 mg protein (samples and positive control), and results were transformed into template copy numbers based on TSR8 standard curves. Mean Table 1. Primer Sequences for RT-PCR.ImmunocytochemistryCells were plated in FNC-coated (AthenaES) 2-well chamber slides and grown to confluence. Cells were carefully washed with PBS and fixed in 4 PFA, 100 220uC-cold methanol, or 100Gene ID hMCT1 NM_003051.Primer Name hMCT1-F hMCT1-RSequence (59?9) AGCGAAGTGTCATGGATATCCTCC CAACACAGCAGTTTAGTAGCAAGC CCCACATGTACACAGAGTATCTGG AGGGTTCTATTCTCTAGCACCAGG GTCAGTGTCTTCTTCAAGGAGCTC AAGTAGCGGTTCAGCATGATGAGC CAGTTCTTTCTCCGAGAGGATGAC TGACTCTTCATGAGGTCTAGGTCG TTGGCATCTGTATTGTGGTGGTGG CAGCTTTGAATTCCTGCTGCTTGG TGGTGATGACAGCCTCTTCTTCAG CTCTGCAAACTTGGAGATGTCCTC TGTCTTGACCTCAATGTGAGCTGC GAAAGTCTCATGCTATCCAGCTGG TTCCACCCATGGCAAATTCCATGG GAGGCATTGCTGATGATCTTGAGGPosition 529?cDNA (bp)hMCT2 NM_004731.3 hMCT2 1317923 NM_004731.3 hMCT4 NM_001042423.1 hMCT4 NM_001042423.1 hAE2 U62531.1 hAE2 U62531.1 hCA12 BC023981.1 hCA12 BC023981.1 hCFTR NM_000492.3 hCFTR NM_000492.3 hsAC10 NM_018417.4 hsAC10 NM_018417.4 hGAPDH NM_002046.3 hGAPDH NM_002046.3 doi:10.1371/journal.pone.11089-65-9 custom synthesis 0051427.thMCT2-F hMCT2-R hMCT4-F hMCT4-R hAE2-F hAE2-R hCA12-F hCA12-R hCFTR-F hCFTR-R hsAC10-F hsAC10-R hGAPDH-F hGAPDH-R2075?419 2075?419 242?40 242?40 631?001 631?001 1071?431 1071?431 1401?737 1401?737 2426?772 2426?772 252?54 252?345 345 299 299 371 371 361 361 337 337 347 347 303Telomerase-Immortalized Human Corneal Endothelium220uC-cold acetone, depending on the primary antibody used. After washing twice with PBS, PFA-fixed cells were permeabilized for 5 min in 0.2 TritonTM X-100 (Sigma-Aldrich) in PBS. Fixed cells were blocked in 1 BSA in PBS for 1 hr at room temperature, before primary antibody was added at 4uC over night. Primary antibodies used were mouse and rabbit anti-ZO-1 (both 1:100; Invitrogen) and mouse anti-Na ATPase a1 (1:100; Abcam, La Jolla, CA). Cells were washed twice with PBS and were incubated with FITC-labeled secondary antibodies (1:100; Jackson ImmunoResearch Laboratories) in blocking buffer fo.Perature. Membranes were incubated overnight on a horizontal shaker at 4uC, with primary antibodies diluted in blocking buffer. Primary antibodies used were collagen type 8 a2 (1:100), N-cadherin (1:200), p53 (1:200), and p16INK4 (1:100) (all Santa Cruz Biotechnology; Santa Cruz, CA), cyclin D (1:500; Millipore), CDK4 (1:200; Thermo ScientificH), Phospho-p53 (1:5000; Cell Signaling Technology; Danvers, MA) and mouse monoclonal b-actin (1:10000, Sigma Aldrich). Blots were rinsed with TBST 3 times for 10 min each and exposed to HRPconjugated donkey anti-mouse or -rabbit IgG for 1 hr at room temperature. All secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA) and diluted 1:2000 in blocking buffer. After washing 3 times in TBST, proteins were detected with SuperSignalH Pico or Femto chemiluminescent substrate (Thermo Scientific). A minimum of 2 different passages were analyzed per group and cell type.Telomeric Repeat Amplification Protocol (TRAP)TRAP assays were performed using the TRAPeze RT kit (Millipore) according to the manufacture’s protocol. Briefly, cells were harvested, washed with PBS, and stored at 280uC until use. After thawing, cells were immediately extracted with CHAPS lysis buffer (Millipore) and total protein concentration was determined using the BCATM Reducing Agent Compatible Assay (Pierce; Rockfort, IL) TRAP reactions were performed with 1 mg protein (samples and positive control), and results were transformed into template copy numbers based on TSR8 standard curves. Mean Table 1. Primer Sequences for RT-PCR.ImmunocytochemistryCells were plated in FNC-coated (AthenaES) 2-well chamber slides and grown to confluence. Cells were carefully washed with PBS and fixed in 4 PFA, 100 220uC-cold methanol, or 100Gene ID hMCT1 NM_003051.Primer Name hMCT1-F hMCT1-RSequence (59?9) AGCGAAGTGTCATGGATATCCTCC CAACACAGCAGTTTAGTAGCAAGC CCCACATGTACACAGAGTATCTGG AGGGTTCTATTCTCTAGCACCAGG GTCAGTGTCTTCTTCAAGGAGCTC AAGTAGCGGTTCAGCATGATGAGC CAGTTCTTTCTCCGAGAGGATGAC TGACTCTTCATGAGGTCTAGGTCG TTGGCATCTGTATTGTGGTGGTGG CAGCTTTGAATTCCTGCTGCTTGG TGGTGATGACAGCCTCTTCTTCAG CTCTGCAAACTTGGAGATGTCCTC TGTCTTGACCTCAATGTGAGCTGC GAAAGTCTCATGCTATCCAGCTGG TTCCACCCATGGCAAATTCCATGG GAGGCATTGCTGATGATCTTGAGGPosition 529?cDNA (bp)hMCT2 NM_004731.3 hMCT2 1317923 NM_004731.3 hMCT4 NM_001042423.1 hMCT4 NM_001042423.1 hAE2 U62531.1 hAE2 U62531.1 hCA12 BC023981.1 hCA12 BC023981.1 hCFTR NM_000492.3 hCFTR NM_000492.3 hsAC10 NM_018417.4 hsAC10 NM_018417.4 hGAPDH NM_002046.3 hGAPDH NM_002046.3 doi:10.1371/journal.pone.0051427.thMCT2-F hMCT2-R hMCT4-F hMCT4-R hAE2-F hAE2-R hCA12-F hCA12-R hCFTR-F hCFTR-R hsAC10-F hsAC10-R hGAPDH-F hGAPDH-R2075?419 2075?419 242?40 242?40 631?001 631?001 1071?431 1071?431 1401?737 1401?737 2426?772 2426?772 252?54 252?345 345 299 299 371 371 361 361 337 337 347 347 303Telomerase-Immortalized Human Corneal Endothelium220uC-cold acetone, depending on the primary antibody used. After washing twice with PBS, PFA-fixed cells were permeabilized for 5 min in 0.2 TritonTM X-100 (Sigma-Aldrich) in PBS. Fixed cells were blocked in 1 BSA in PBS for 1 hr at room temperature, before primary antibody was added at 4uC over night. Primary antibodies used were mouse and rabbit anti-ZO-1 (both 1:100; Invitrogen) and mouse anti-Na ATPase a1 (1:100; Abcam, La Jolla, CA). Cells were washed twice with PBS and were incubated with FITC-labeled secondary antibodies (1:100; Jackson ImmunoResearch Laboratories) in blocking buffer fo.
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