Typhoon fla 7000 imaging system

Cell lysis was performed using NP-40 buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl and 1% NP-40) supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Protein denaturation was achieved after diluting (1:1) the samples with 2× Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, CA, USA), followed by heating at 95 °C for 5 min. The samples were analyzed with SDS-PAGE, and proteins were electrophoretically transferred to PVDF membrane for Western blot analysis. Membranes were treated with blocking solution, consisting of 5% non-fat dry milk in PBST, for 1 h at RT. For the detection of CYLD, we used the cylindromatosis 1 antibody (E-4, sc-74434, Santa Cruz Biotechnology, Dallas, TX, USA), and for b-actin, we used the beta actin antibody (C-4, Santa-Cruz). As a secondary antibody, we used m-IgGκ BP-HRP (sc-516102, Santa Cruz Biotechnology, Dallas, TX, USA) for both CYLD and b-actin. In all cases, the antibodies were diluted in blocking solution, and the membrane was treated for 1 h at RT. Membrane-bound antibodies were detected with an enhanced chemiluminescence detection kit (Pierce, Waltham, MA, USA) using a Typhoon FLA 7000 imaging system (GE Healthcare Life Sciences, Chicago, IL, USA). The E-4 CYLD antibody interacts with the C-terminal 419 amino acids of human CYLD.

Kinase and phosphatase assays were adapted from reference 33 (link). Unless otherwise stated, 5 µM protein concentrations were used. Reaction mixtures were incubated in dialysis buffer in the presence of 500 µM ATP and 2.5 µCi [γ-³²P]ATP (3,000 Ci mmol⁻¹; Hartmann Analytic) at room temperature. Additional proteins were added, and reactions were stopped by the addition of SDS sample buffer at indicated time points. Reaction mixtures were stored on ice or loaded on 12% SDS gels. Wet gels were exposed to phosphor screens (0.5 to 1.5 h) before being scanned using a Typhoon FLA7000 imaging system (GE Healthcare). In experiments assessing phosphatase activity, ATP was depleted by the addition of 1.5 units of hexokinase (Roche) and 5 mM d-glucose 15 min after phosphorylation. For the purification of MrrA~P, the following conditions were used. CC2874 (0.2 µM) and MrrA (100 µM) were prephosphorylated for 1 h. Twenty-five microliters of anti-MBP magnetic beads (New England Biolabs; E8037S) was added and incubated for 1 h. Beads were then concentrated using a magnet. hexokinase and glucose were added to the supernatant as described above and incubated for 10 min to deplete remaining ATP.

To probe potential SecYEG:FtsQ contacts, 100 nM RNC FtsQ variants bearing single cysteines (mutations V40C, S41C, G42C, and W43C) in the FtsQ TMH were incubated with ~1 μM SecYEG‐ND, which contained single cysteines within the translocon lateral gate (M83C, S87C, I282C, and I283C). After 15‐min incubation at the ambient temperature, cupper phenanthroline was added to the concentration of 1 mM, and the cross‐linking reaction was conducted for 30 min at the ambient temperature. Cross‐linking products containing the nascent chain were detected via Western blotting 75. Western blots were developed using ECL Western blotting substrate (Pierce) and imaged using LAS‐4000 Mini imager (GE Life Sciences). To detect SecYEG‐based cross‐linking products, the cysteines within the lateral gate were combined with a cysteine at the translocon periplasmic interface (mutation L148C), which was labeled with CF488A‐maleimide (Sigma/Merck), as previously described 29. For the cross‐linking experiments, 100 nM SecY^CF488AEG‐ND variants was mixed with 200 nM non‐translating ribosomes or RNCs, and the cross‐linking with copper phenanthroline was conducted as described above. Where indicated, samples were treated with N‐ethylmaleimide prior loading on SDS–PAGE. In‐gel fluorescence was recorded using Typhoon FLA 7000 imaging system (GE Life Sciences).

Telomerase activity was measured as previously described (35 ). In brief, 0.5 × 10⁶ cells were collected for each sample and lysed at 1 × 10⁶ cells /100 μl in CHAPS lysis solution supplemented with 0.1 mM PMSF. 10,000 cell equivalents of CHAPS lysate were used for telomere synthesis in a 10 μl volume containing the following components: 1X Telomerase reaction buffer (50 mM Tris-OAc at pH 8.5, 1 mM MgCl₂, 50 mM KOAc, 5 mM β-mercaptoethanol, 10 mM spermidine), 2 mM telomere dNTP mix (dATP, dGTP, dTTP), and 1 μM of PAGE-purified Ts oligo. Two microliter of telomere synthesis solution were used in 15 μl PCR reactions including 1.2 units of Platinum Taq, 1.5 mM MgCl₂, 0.2 mM dNTP, 200 μM TAMRA-TS, NT, ACX primers, and 1 amole TSNT oligo as an internal quantification control. PCR cycling conditions were as follows: 94°C for 2 min, followed by 32 cycles of 94°C 15 s, 60°C for 20 s. TRAP PCR products were run on non-denaturing 1X TBE buffered 12% PAGE gels and fluorescent TAMRA-TS amplified telomerase products were detected in the Cy3 channel on a Typhoon FLA7000 imaging system (GE Healthcare Life Sciences). Telomerase activity was quantified in ImageJ as the ratio of the first 8 telomerase products to the TSNT internal control. The same lot of Jurkat T cell lysate was used for normalization among different batches of TRAP results.

Telomerase activity was measured as previously described (21 ). In brief, 0.5×10⁶ cellswere collected for each sample and lysed at 1× 10⁶cells/100 μl in CHAPS lysis solution supplemented with 0.1mM PMSF. Ten thousand cell equivalents of CHAPS lysate were used for telomere synthesis in a 10-μl volume containing the following components: 1× telomerase reaction buffer (50 mM Tris-OAc [pH 8.5], 1 mM MgCl₂, 50 mM KOAc, 5 mM 2-ME, and 10 mM spermidine), 2 mM telomere dNTP mixture (dATP, dGTP, dTTP), and 1 mM of PAGE-purified Ts oligonucleotide. Two microliters of telomere synthesis solution were used in 15-μl PCRs, including 1.2 U of Platinum Taq, 1.5 μM MgCl₂, 0.2 μM dNTP, 200 μM TAMRA-TS, NT, and ACX primers, and 0.1–1-amole TSNT oligonucleotide. PCR cycling conditions were as follows: 94°C for 2 min, followed by 32–35 cycles of 94°C 15 s and 60°C for 20 s. The PCR products were separated on nondenaturing 12% PAGE gels and fluorescent TAMRA-TS–amplified telomerase products were detected in the Cy3 channel on a Typhoon FLA7000 imaging system (GE Healthcare Life Sciences). Telomerase activity was quantified in ImageQuant or ImageJ as the ratio of the first six telomerase products to the TSNT internal control.