Peptide amount: 1 ng, 4 ng, 16 ng, 64 ng
Blocking buffer: 5% NFDM/TBST
Primary Ab dilution: 1:2000
Primary Ab incubation condition: 2 hours at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 60 seconds
The list of peptides used in the experiment is provided below.
Lane 1: H3K23 acetyl. Dot 2: H3K23 crotonyl.
Dot 3: H3K23 propionyl. Dot 4: H3K23 butyryl.
Dot 5: H3K23 β-hydroxybutyryl. Dot 6: H3K23 2-hydroxyisobutyryl.
Dot 7: H3K18 acetyl. Dot 8: H3K23 unmodified.

Lysates: (-) HeLa cells; (+) HeLa cells treated with 30 mM sodium butyrate for 4 hours
Protein loading amount: 20 μg
Blocking buffer: 5% NFDM/TBST
Primary Ab dilution: 1:20000
Primary Ab incubation: 2 hours at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 3 seconds
Predicted band size: 15 kDa
Observed band size: 15 kDa

Lysates: (-) HeLa cells; (+) HeLa cells treated with 5 mM sodium butyrate for 24 hours
Protein loading amount: 20 μg
Blocking buffer: 5% NFDM/TBST
Primary Ab dilution: 1:20000
Primary Ab incubation: 2 hours at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 3 seconds
Predicted band size: 15 kDa
Observed band size: 15 kDa

Lysates: (-) NIH-3T3 cells; (+) NIH-3T3 cells treated with 30 mM sodium butyrate for 4 hours
Protein loading amount: 20 μg
Blocking buffer: 5% NFDM/TBST
Primary Ab dilution: 1:20000
Primary Ab incubation: 2 hours at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 10 seconds
Predicted band size: 15 kDa
Observed band size: 15 kDa

Lysates: (-) C6 cells; (+) c6 cells treated with 5 mM sodium butyrate for 24 hours
Protein loading amount: 20 μg
Blocking buffer: 5% NFDM/TBST
Primary Ab dilution: 1:5000
Primary Ab incubation: 2 hours at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 30 seconds
Predicted band size: 15 kDa
Observed band size: 15 kDa

Tissue: Human testis
Section type: Formalin-fixed & paraffin-embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0
Primary Ab dilution: 1:1000
Primary Ab incubation: 1 hour at room temperature
Secondary Ab: Anti-Rabbit and Mouse Polymer HRP (Ready to Use)
Counter stain: Hematoxylin (blue)
Description: The brown color represents the positive signal observed with PTM-115RM.

Tissue: Human placenta
Section type: Formalin-fixed & paraffin-embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0
Primary Ab dilution: 1:1000
Primary Ab incubation: 1 hour at room temperature
Secondary Ab: Anti-Rabbit and Mouse Polymer HRP (Ready to Use)
Counter stain: Hematoxylin (blue)
Description: The brown color represents the positive signal observed with PTM-115RM.

Tissue: Human bladder carcinoma
Section type: Formalin-fixed & paraffin-embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0
Primary Ab dilution: 1:1000
Primary Ab incubation: 1 hour at room temperature
Secondary Ab: Anti-Rabbit and Mouse Polymer HRP (Ready to Use)
Counter stain: Hematoxylin (blue)
Description: The brown color represents the positive signal observed with PTM-115RM.

Tissue: Mouse cerebrum
Section type: Formalin-fixed & paraffin-embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0
Primary Ab dilution: 1:1000
Primary Ab incubation: 1 hour at room temperature
Secondary Ab: Anti-Rabbit and Mouse Polymer HRP (Ready to Use)
Counter stain: Hematoxylin (blue)
Description: The brown color represents the positive signal observed with PTM-115RM.

Tissue: Rat cerebrum
Section type: Formalin-fixed & paraffin-embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0
Primary Ab dilution: 1:1000
Primary Ab incubation: 1 hour at room temperature
Secondary Ab: Anti-Rabbit and Mouse Polymer HRP (Ready to Use)
Counter stain: Hematoxylin (blue)
Description: The brown color represents the positive signal observed with PTM-115RM.

IP of whole cell lysates from HeLa cells treated with 5 mM sodium butyrate for 24 hours
Lane 1: 5% Input
Lane 2: IP with PTM-115RM
Lane 3: IP with Rabbit mAb IgG Isotype Control
IP Ab incubation: 1:100 dilution, 4°C overnight
Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
WB Primary Ab incubation: PTM-115RM, 1:1000 dilution, 2 hours at room temperature
Secondary Ab: Goat Anti-Rabbit IgG for IP (HRP Conjugate)
Exposure time: 120 seconds
Predicted band size: 15 kDa
Observed band size: 15 kDa

Sample: HeLa cells treated with 5 mM sodium butyrate for 24 hours
Cross-linking conditions: no cross-linking
Amount of chromatin per IP: 100 μg
Amount of Ab per IP: 6 μg
Beads type and amount per IP: 50 μL of Protein A MagBeads
Description: Chromatin immunoprecipitations were performed with 6 μg of normal rabbit IgG as a negative control. The immunoprecipitated DNA was quantified by real-time PCR using primers specific for the human GAPDH-CDS, RPL30, LDHA-promoter, LDHA-CDS, FOXO3a-promoter, FOXO3a-downstream, RAB20, and TUBBP10 regions. The data are presented as enrichment of each sample relative to the total amount of input chromatin at each amplicon.