Mncl2

SV40T immortalized tetraploid female Rosa26-M2rtTA MEFs were passaged asynchronously, harvested, PBS washed and aliquoted 4 × 10⁶ cells/tube. Cell pellets were then flash frozen in liquid nitrogen. Pellets were thawed and then sonicated on ice in 1x NEBuffer Pack for Protein MetalloPhosphatases with 1 mM MnCl₂ (New England Biolabs) and freshly added 1 mM PMSF, 1 μg/mL pepstatin and 1 μg/mL leupeptin. 1 × 10⁶ cell equivalents were treated with 1000U of Lambda Protein Phosphatase (New England Biolabs) at 30C for 15, 30, or 60 min. Lambda Protein Phosphatase was excluded from the negative control sample which was incubated at 30°C for 60 min in buffer supplemented with 10 mM sodium pyrophosphate, 10 mM B-glycerophosphate, 5 mM NaF, 1 mM Na₃VO₄. Reactions were stopped with an equal volume of 2x Laemmli sample buffer and heated at 95°C for 4 min. Samples were analyzed by SDS-PAGE and western blot.

Locust brains (10–12 individuals/sample) were collected and homogenized in 1 X PBS buffer (0.1 M phosphate buffer, 0.15 M NaCl, pH 7.4) containing the phosphatase inhibitor PhosSTOP (Roche) and a proteinase inhibitor (CoWin). Total protein content was examined using the BicinChoninic Acid (BCA) Protein Assay Kit (Thermo). The extracts (100 μg) were reduced, denatured, and electrophoresed on 8% SDS-PAGE gel and then transferred to polyvinylidene difluoride membrane (Millipore). The membrane was then cut to two pieces and incubated separately with a specific antibody against the target protein of ~130 KD or a reference protein of ~55 KD overnight at 4°C (affinity-purified polyclonal rabbit antibody against uNOS, Sigma-Aldrich, 1:200; Rabbit polyclonal antibody against tubulin, CoWin, 1:5000). Goat anti-rabbit IgG was used as secondary antibody (CoWin, 1:10000). Protein bands were detected by chemiluminescence (ECL kit, CoWin). The band intensity of the Western blot was quantified using densitometry in Quantity One software.
For determination of NOS phosphorylation, 200 μg brain extracts were incubated with λ phosphatase and 1 X NEB buffer supplemented with 1 mM Mncl₂ for 1 hr at 30°C (NEB). Control protein was treated under the same conditions without λ phosphatase. Western blot analysis was performed to confirm NOS phosphorylation in the locust brains.

To check protein expression, cells were induced by the addition of doxycycline (10 ng/ml) for 24 hours, and 30 μg of total cell lysate was analyzed by Western blotting with a PrimPol antibody in comparison to α-tubulin controls. To look specifically at PrimPol phosphorylation, a phospho-peptide antibody was generated (Eurogentec). Antibodies were raised in rabbits to the peptide ac- ELAEAAEN-S(PO3H2)-LLS+C –conh2 and affinity-purified. The specificity of the antibody was confirmed by Western blotting of phosphatase-treated cell lysate. Briefly, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer. Five microliters of PMP buffer (New England Biolabs) and MnCl₂ (New England Biolabs) was then added to 40 μl of protein sample and then incubated at 30°C for 1 hour with or without 400 U of λ phosphatase. The specificity of the antibody was assessed by Western blot.

To determine SCOC phosphorylation in vivo (Fig. 3C), myc-ULK1 WT or myc-ULK1 KI were co-expressed along with pEGFPC1, pEGFPC1-SCOC or pEGFPC1-SCOC 9A in HEK293A cells. 24 h after transfection, cells were starved for 2 h with EBSS. Cells were then harvested and washed with cold PBS, and lysed in TNTE buffer (w/o 10% v/v glycerol). The cell lysates were immunoprecipitated using GFP-TRAP (ChromoTek) as decribed above. Prior to lambda-phosphatase treatment, GFP-TRAP beads were washed 3 times with TNTE buffer (w/o 10% v/v glycerol, EDTA and PhosSTOP) and split into two equal portions for λ-phosphatase treatment and control. Lambda phosphatase was added into each sample with phosphatase reaction buffer and MnCl₂ following manufacturer’s instructions (New England BioLabs). Sodium orthovanadate (Na₃VO₄) was added into each sample at the final concentration of 100 μM, as a control. All reactions were incubated at 30 °C for 30 min. 2x Laemmli buffer was added into all the samples to stop reaction and then boiled at 100 °C for 5 min. Samples were separated by 10% SDS-PAGE with 25 μM Phos binding reagent (Phosbind) acrylamide (APExBIO, F4002) and analysed by immunoblotting.

Microcystin-LR was purchased from Sigma (Saint-Quentin Fallavier, France). PP2A was obtained from New England Biolabs Inc. Na₂S₂O₃, MgCl₂, MnCl₂, HCl, Ca(ClO)₂, high-purity CO₂, p-Nitrophenyldisodium orthophorphate (p-NPP), tris(hydroxymethyl)aminomethane (Tris), bovine serum albumin (BSA), dithiothreitol (DTT), neoprene rubber, sodium nitrobenzene disodium, and ascorbic acid were purchased from Sinopharm (Shanghai, China). HCOOH, CH₃OH, CF₃COOH, and HPLC acetonitrile were purchased from Merck (Darmstadt, Germany).

Cell lysates (39 µL), prepared as above, were mixed with 5 µL MnCl₂ (New England Biolabs) and 5 µL 10× protein metallophosphatases (PMP) buffer (New England Biolabs). Samples that were lysed in the absence of NaF also received 1 µL lambda protein phosphatase (LPP; New England Biolabs). After incubation at 30 °C for 30 min, 6× SDS loading buffer was added, and samples were analyzed by Phos-tag PAGE.