Peptide amount: 1 ng, 4 ng, 16 ng, 64 ng
Blocking buffer: 5% NFDM/TBST
Primary Ab dilution: 1:2000
Primary Ab incubation: 2 hours at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 30 seconds
The list of peptides used in the experiment is provided below.
Lane 1: H3K23 L-lactyl. Lane 2: H3K18 L-lactyl.
Dot 3: H3K23 acetyl. Dot 4: H3K23 crotonyl.
Dot 5: H3K23 butyryl. Dot 6: H3K23 protonyl.
Dot 7: H3K23 unmodified.

Lysates: (-) HeLa cells; (+) HeLa cells treated with 100 mM sodium lactate for 24 hours
Protein loading amount: 20 μg
Blocking buffer: 2.5% BSA/TBST
Primary Ab dilution: 1:10000 (2.5% BSA)
Primary Ab incubation: overnight at 4°C
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
Important Note: It is recommended to employ 2.5% BSA/TBST for blocking and antibody dilution. Using 5% nonfat milk may result in nonspecific bands.

Lysates: (-) NIH-3T3 cells; (+) NIH-3T3 cells treated with 25 mM sodium lactate for 24 hours
Protein loading amount: 20 μg
Blocking buffer: 2.5% BSA/TBST
Primary Ab dilution: 1:10000 (2.5% BSA)
Primary Ab incubation: overnight at 4°C
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
Important Note: It is recommended to employ 2.5% BSA/TBST for blocking and antibody dilution. Using 5% nonfat milk may result in nonspecific bands.

Lysates: (-) C6 cells; (+) C6 cells treated with 25 mM sodium lactate for 24 hours
Protein loading amount: 20 μg
Blocking buffer: 2.5% BSA/TBST
Primary Ab dilution: 1:10000 (2.5% BSA)
Primary Ab incubation: overnight at 4°C
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
Important Note: It is recommended to employ 2.5% BSA/TBST for blocking and antibody dilution. Using 5% nonfat milk may result in nonspecific bands.

Tissue: human colon
Section type: formalin fixed & paraffin-embedded section
Antigen retrieval method: high temperature and high pressure
Antigen 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)
Counterstain: hematoxylin (blue)
Description: The brown color represents the positive signal observed with PTM-1413RM.

Tissue: human placenta
Section type: formalin fixed & paraffin-embedded section
Antigen retrieval method: high temperature and high pressure
Antigen 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)
Counterstain: hematoxylin (blue)
Description: The brown color represents the positive signal observed with PTM-1413RM.

Sample: HeLa cells treated with 25 mM sodium lactate for 24 hours
Cross-linking conditions: no cross-linking
Amount of chromatin per IP: 5×10⁶ cells
Amount of Ab per IP: 6 μg
Bead 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 RPL30, LDHA promoter, FOXO3a promoter, FOXO3a downstream, RAB20, and TUBBP10 regions. The data are presented as enrichment of each sample relative to the total amount of input at each amplicon.