Peptide amount: 4 ng, 20 ng, 100 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: 15 seconds
The list of peptides used in the experiment is provided below.
Lane 1: L-lactylated peptide library. Lane 2: acetylated peptide library.
Lane 3: propionylated peptide library. Lane 4: butyrylated peptide library.
Lane 5: crotonylated peptide library. Lane 6: malonylated peptide library.
Lane 7: 2-hydroxyisobutyrylated peptide library. Lane 8: succinylated peptide library.
Lane 9: unmodified peptide library.

Peptide amount: 10 ng, 20 ng, 50 ng, 100 ng, 500 ng
Blocking buffer: 3% BSA/TBST
Primary Ab dilution: 1:1500
Primary Ab incubation condition: 1 hour at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 15 seconds
The list of peptides used in the experiment is provided below.
Kce: carboxyethylated peptide library. KD-la: D-lactylated peptide library.
KL-la: L-lactylated peptide library. Kac: acetylated peptide library.
K: Unmodified peptide library.

Peptide amount: 10 ng, 20 ng, 50 ng, 100 ng, 500 ng
Blocking buffer: 3% BSA/TBST
Primary Ab dilution: 1:1500
Primary Ab incubation condition: 1 hour at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 15 seconds
The peptides used in the experiment are synthetic, sequence-specific histone
peptides with the following modifications:
KL-la: L-lactylated peptide library. KD-la: D-lactylated peptide library.
Kce: carboxyethylated peptide library.

Samples: BSA, Acetylated BSA, L-lactylated BSA, D-lactylated BSA, carboxyethylated BSA
Blocking buffer: 3% BSA/TBST
Primary Ab dilution: 1:1500
Primary Ab incubation condition: 1 hour at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 15 seconds

Lysates: MCF-7 cells treated with 0.1, 1, 5, and 25 mM glucose
Blocking buffer: 3% BSA/TBST
Primary Ab dilution: 1:1500
Primary Ab incubation condition: 1 hour at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 30 seconds
Predicted MW: Multiple
Observed MW: Multiple

Lysates: (-): HeLa cells; (+): HeLa cells treated with 100 mM sodium lactate for 24 hours
Protein loading amount: 20 μg
Blocking buffer: 5% NFDM/TBST
Primary Ab dilution: 1:1000
Primary Ab incubation condition: 2 hours at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 30 seconds
Predicted band size: Multiple
Observed band size: Multiple

Tissue: Human colon cancer
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 condition: 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-1425RM

Tissue: Mouse intestine
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 condition: 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-1425RM

Samples: (A) HeLa cells; (B) HeLa cells treated with 100 mM sodium lactate for 24 hours
Fixative: 100% Ice-cold methanol
Permeabilization: 0.1% Triton X-100
Primary Ab dilution: 1:100
Primary Ab incubation condition: 4°C overnight
Secondary Ab: Goat Anti-Rabbit IgG
Nuclear counter stain: DAPI (blue)
Counter stain: Tubulin (red)
Description: The green color represents the positive signal observed with PTM-1425RM.

Sample: HeLa cells
Fixative: 4% Paraformaldehyde
Permeabilization: 0.1% Triton X-100
Primary Ab dilution: 1:100
Secondary Ab: Goat Anti-Rabbit IgG
Unlabeled control: The cell without incubation with primary antibody and secondary antibody (black line).
Isotype control: Rabbit mAb IgG Isotype Control (blue line).
Description: The red line represents the positive signal observed with PTM-1425RM.

IP of HeLa + sodium lactate (100 mM, 24 hours) extracts
Lane 1: 5% Input
Lane 2: IP with PTM-1425RM
Lane 3: IP with Rabbit mAb IgG Isotype Control
IP Ab incubation condition: 4°C overnight, 1:100 dilution
Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
WB primary Ab incubation condition: PTM-1425RM, 2 hours at room temperature, 1:1000 dilution
Secondary Ab: Anti-Rabbit IgG for IP (HRP Conjugate)
Exposure time: 60 seconds
Predicted band size: Multiple
Observed band size: Multiple

Sample: MCF-7 cells
Cross-linking conditions: no cross-linking
Amount of chromatin per IP: 5×10⁶ cells
Amount of Ab per IP: 6 μg
Beads type and amount per IP: 50 μL of Protein A/G 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 LDHA CDS, FOXO3a downstream, and TUBBP10 regions. The data are presented as enrichment of each sample relative to total amount of input at each amplicon.