*4.4. Immunoprecipitation*

Cells were lysed in lysis bu ffer (150 mM NaCl, 20 mM Tris, pH7.4, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL pepstatin,1 mM Na3VO4, and 2.5 mM β-glycerophosphate). Cell lysates (1 mg) were incubated with 1 μL α-Flag antibodies (Sigma-Aldrich, F4042) in lysis bu ffer containing 0.2% Triton X-100 for 3 h at 4 ◦C followed by incubation with 20 μL protein G agarose-beads (GE Healthcare) for 1–3 hours at 4 ◦C. At the end of incubation, beads were washed four times with lysis bu ffer without Triton X-100. The phosphorylation states of p38 α and the potential associated proteins were detected by Western Blotting. Alternatively, the immunoprecipitates were fractionated by SDS-PAGE and subjected to mass spectrometric analysis to examine the interactome.

To immunoprecipitate HA-PRMTs for methylation assay, cell lysates (0.5 mg) were incubated with α-HA antibodies (0.5 μL) (BioLegend, #901501) as described above and followed by incubation with 10 μL protein G agarose-beads (GE Healthcare). At the end of incubation, beads were washed twice with lysis bu ffer without Triton X-100, twice with PBS contained 0.2% tween-20, then twice with 25 mM Tris-HCl pH7.9. The immunoprecipitated HA-PRMTs were used immediately as an enzyme source for in vitro methylation assay.

#### *4.5. In Vitro Methylation Assay*

For the mass spectrometric identification of methylation sites in p38 α, the methylation assay was carried out in vitro using recombinant wild-type His-p38 α proteins as a substrate and with or without recombinant GST-PRMT1 proteins as an enzyme. The reactions were carried out using S-adenosyl-methionine (AdoMet, PerkinElmer) as a methyl donor as described previously [20]. Reactions were terminated and subjected to SDS-PAGE fractionation. The protein band containing p38 α was excised from the gel and subjected to mass spectrometric analysis to examine its posttranslational modifications.

To compare methyl incorporation into the wild-type and mutant His-p38 α proteins, HA-PRMT1, HA-PRMT1G80R, HA-PRMT6, HA-PRMT6KA proteins were expressed from the pcDNA3-HA2 plasmids in K562 cells and immunoprecipitated using anti-HA antibodies. Methylation reactions were carried out using S-adenosyl-L-[methyl-3H]methionine (3H-AdoMet, 1.65 μCi, PerkinElmer) as a methyl donor. Methyl incorporation was visualized by fluorography as described [20].

#### *4.6. Mass Spectrometric Analysis*

The gel pieces from SDS-PAGE were minced and incubated with trypsin (Promega). Peptides were extracted from the gel pieces by sonication in 50% acetonitrile containing 0.1% formic acid. The solutions were dried. The peptides were resuspended in 0.1% formic acid and analyzed by nanoflow high-performance liquid chromatography (Agilent Technologies 1200 series) followed by a LTQ-Orbitrap Discovery hybrid mass spectrometer with a nano-eletrospray ion source (Thermoelectron). This was performed by the Proteomics Center of National Yang- Ming University. The raw data were processed by the Xcalibur 2.0 SR1 software (Thermoelectron) and were converted to DTA files. Protein identity was determined by comparing the peptide sequences against the UniProtKB protein database (http://www.uniprot.org/) with TurboSequest search server T (version 27, revision 11) [36]. A protein was identified when two peptide ions matched with an Xcorr score >2.5. For the identification of p38 α methylation sites, post-translational modifications (PTMs) of the peptides were identified by the in-house PTM finder program [37]. The peptides that contained PTM were matched with an Xcorr score >2.0. For the identification of associated proteins, quantitative analysis with MS spectra counting was performed by an in-house tool within a Microsoft VBA environment. MS spectra counts were normalized to total identified spectra per sample and then normalized to p38 α spectra counts.

#### *4.7. Limited Trypsin Digestion of Recombinant p38*α

The recombinant His-tagged p38 α WT and mutants (R49K, R149K and R49/149K) proteins (5 μg) were incubated with 0.5 μg trypsin (Sigma) in a final volume of 20 μL for 5, 15, and 30 min at 37 ◦C. Samples were analyzed by SDS-PAGE.

## *4.8. Erythroid Di*ff*erentiation*

For erythroid di fferentiation, K562 cells were treated with 1-beta-D-arabinofuranosylcytosine (AraC, 1μM, Sigma-Aldrich) for various times as indicated. Hemoglobin production was detected by using a benzidine/hydrogen peroxide solution as described [20]. Stained cells were counted under a light microscope. Two hundred cells were examined in each assay.

#### *4.9. Real-Time Reverse Transcription PCR*

Total RNA was extracted by using an illustra RNAspin Mini Isolation Kit (GE Healthcare). Isolated RNAs (4 μg) were subjected to cDNA synthesis with a RevertAid first strand cDNA synthesis kit (Fermentas). Real-time PCR was performed on an ABI StepOne Plus Real-Time PCR System (Applied Biosystems) using SYBR-Green reagents (SensiFAST SYBR Hi-ROX mix, Bioline). The samples were examined in triplicate for each assay and analyzed by using the comparative

cycle threshold CT (ΔΔ CT) method [3]. The expression of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control for cDNA contents. Gene-specific amplification was conducted with the following primer sets: for EKLF, 5'-CGGCAAGAGCTACACCAAG-3' (sense) and 5'-CCGTGTGTTTCCGGTAGTG-3' (antisense); for GATA1, 5'-CAGTCTTTCAGGTGTACCC -3' (sense) and 5'-GAGTGATGAAGGCAGTGCAG-3' (antisense); for ALAS2, 5'-GCAGCACT CAACAGCAAG-3' (sense) and 5'-ACAGGACGGCGACAGAAA-3' (antisense); for PBGD, 5'-CGCCTCCCTCTAGTCTCTGCTTCT-3' (sense) and 5'- GTTGCCACCACACTGTCCGTCTG-3' (antisense); for GAPDH, 5'-TGGTATCGTGGAAGGACTCATGAC-3' (sense) and 5'-ATGCCA GTGAGCTTCCCGTTCAGC-3' (antisense).

## *4.10. Western Blotting*

Cells were lysed in RIPA bu ffer (150 mM NaCl, 10 mM Tris, pH7.4, 0.1% SDS, 1% Triton X-100, 5 mM EDTA, 1% Na-deoxycholate, 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL pepstatin). The primary antibodies used were α-Flag (Sigma-Aldrich, F4042, 1:2000 or Sigma-Aldrich, F7425, 1:1000), α-HA (BioLegend, #901501, 1:1000), α-p38 (Cell signaling, #9212, 1:1000), α-pp38 (Cell signaling, #9219, 1:1000), α-MKK3 (Cell signaling, #5674, 1:500), α-MKK6 (Abnova, #M02, 1:500), α-MAPKAPK2 (Cell signaling, #12155, 1:1000), α-PRMT1 (Sigma-Aldrich, #P16220, 1:1000), α-GAPDH (Cell signaling, G9545, 1:10000), α-mono- and di-methyl arginine (abcam, ab412, 1:500). The secondary antibodies used were anti-mouse (Sigma-Aldrich) or anti-rabbit (Genetex) IgG linked-horseradish peroxidase.
