Typhoon system

Immunoblotting was performed as described (Yang et al. 2020 (link)). The following antibodies were used: mTRF1 (#1449), mTRF2 (#1254), mXPB (Bethyl Laboratories A301-337A), mXPD (CST 11963), mZRANB3 (Abclonal A9555), mPOLD3 (Proteintech 21935-1-AP), mMNAT1 (Proteintech 11719-1-AP), mCDK7 (Proteintech 27027-1-AP), mCCNH (Proteintech 67065-1-Ig), mXPA (Proteintech 16462-1-AP), mCHK1 (Santa Cruz Biotechnology sc-8408), phospho-mCHK1 S345 (CST 2348), mGAPDH (Thermo Fisher MA5-15738), and γ-Tubulin (Sigma-Aldrich GTU88).
For quantitative fluorescent immunoblotting, membranes were blocked with Intercept TBS blocking buffer (Li-Cor 927-60001) for 1 h and then incubated with primary antibodies for 2 h. After three TBST washes, membranes were incubated with fluorescently labeled secondary antibodies IRDye 800CW goat antirabbit (Li-Cor 925-32211) and IRDye 680RD goat antimouse (Li-Cor 925-68070) for 1 h, followed by three TBST washes. Imaging was performed with a GE Typhoon system and quantified using ImageJ (1.51j8).

Proteins in phosphoprotein-enriched fractions were precipitated with a 2-D Clean-Up kit (GE Healthcare; http://www.gelifesciences.com/). Precipitates were dissolved in three equal volumes of precipitant buffer provided by the manufacturer, and the mixture was put on ice for 15 min. The precipitate was recovered by centrifugation and dissolved in the same volume of co-precipitant, and the precipitate was again recovered by centrifugation. The precipitate was dissolved in 25 µl of water, and 1 ml of wash buffer and 5 µl of wash additive were added to the sample. After incubation at –20°C for 30 min, the precipitate was recovered, air-dried and dissolved in buffer that contained 30 mM Tris–HCl (pH 8.5), 2 M thiourea, 7 M urea and 4% CHAPS. Phosphorylated proteins were labeled with the CyDye™ DIGE Fluor Minimal Labeling kit (GE Healthcare) with Cy2, Cy3 or Cy5 according to the manufacturer’s protocol: 400 pmol of dye was added to 50 µg of protein in the above buffer and the mixture was kept on ice for 30 min. A mixture of labeled proteins from wild-type and mutant plants was then subjected to isoelectric focusing with Immobiline™ DryStrip and Ettan IPGphor II (GE Healthcare). After subsequent SDS–PAGE, protein spots were visualized with the Typhoon system (GE Healthcare), and the intensity of each spot was determined with DeCyder software (GE Healthcare).

TRF analysis was conducted as described previously (Celli and de Lange 2005 (link)). Briefly, 1 million cells were embedded in 1% agarose plugs in PBS, digested with 1 mg/mL proteinase K in digestion buffer (100 mM EDTA, 0.2% sodium deoxycholate, 1% sodium lauryl sarcosine), and washed extensively in TE buffer (10 mM Tris at pH 8, 1 mM EDTA). The DNA in the plugs was digested with 60 U of AluI and MboI overnight. Plugs were run on a 1% agarose gel using pulse field gel electrophoresis, and the gel was dried. ³²P-γ-ATP-labeled TelC probes ([CCCTAA]₄) were used for probing telomeric overhang under native conditions. After washing and imaging, the gel was denatured, reprobed, and imaged again. Imaging was performed with a GE Typhoon system.

Northern blot for detection of TERRA was adapted from a previous study (Azzalin et al. 2007 (link)). Briefly, total RNA was extracted using TRIzol. Five micrograms of RNA was run on a 1.2% agarose gel in FA buffer (20 mM MOPS at pH 7.0, 5 mM sodium acetate, 1 mM EDTA, 0.67% formaldehyde). After electrophoresis, the gel was soaked in 0.05 M NaOH and 1.5 M NaCl for 10 min and then in 10× SSC for 15 min. The RNA was then transferred onto a nylon N⁺ membrane and cross-linked by UV. The membrane was blocked in Church buffer (50% sodium phosphate buffer [0.684 M Na₂HPO₄, 0.316 M NaH₂PO₄ at pH 7.2], 1 mM EDTA, 7% SDS, 1% BSA) for 90 min at 55°C followed by hybridization overnight at 60°C with ³²P-γ-ATP-labeled TelC probes ([CCCTAA]₄). The membrane was then washed twice with 2× SSC and 0.2% SDS and once with 0.5× SSC and 0.2% SDS at 55°C, and finally exposed to a PhosphorImager screen and scanned using the GE Typhoon system.

TRF analysis was conducted as described previously (Celli and de Lange 2005 (link)). Briefly, 1 million cells were embedded in 1% agarose plugs and treated with 1 μg/mL proteinase K overnight, followed by extensive wash with TE, after which the plugs were digested with 60 U of AluI and MboI overnight. Plugs were run out in a 1% agarose gel using pulse field gel electrophoresis and the gel was dried. [γ-32P]ATP-labeled TelC probe ([CCCTAA]₄) was used for probing telomeric overhang under native condition overnight. After washing and imaging, the gel was denatured, reprobed, and imaged again. Imaging was performed with a GE Typhoon system.