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.
K_ce: Carboxyethylated peptide library. K_D-la: D-lactylated peptide library.
K_L-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:
K_L-la: L-lactylated peptide library. K_D-la: D-lactylated peptide library.
K_ce: 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: 1:100
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-1401RM.

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: 1:100
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-1401RM.

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-1401RM.

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-1401RM.

IP of HeLa + sodium lactate (100 mM, 24 hours) extracts
Lane 1: 5% Input
Lane 2: IP with PTM-1401RM
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-1401RM, 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.