Figure 1: IL-1 beta target protein structure.

IL-1 beta Introduction

Protein Function

• IL-1 beta is produced and released by various types of immune and non-immune cells in rapid response to inflammatory signals.
• The biosynthesis of most IL-1 family members differs from other cytokines in that they are produced as inactive enzyme precursors and require proteolytic processing to active. In myeloid cells, the N-terminal processing of IL-1 beta (and IL-18) is completed by the inflammasome effector component caspases.
• The IL1B production occurs in 2 steps, each being controlled by different stimuli. First, inflammatory signals, such as LPS, stimulate the synthesis and promote the accumulation of cytosolic stores of pro-IL1B (priming). Then additional signals are required for inflammasome assembly, leading to CASP1 activation, pro-IL1B processing and eventually secretion of the active cytokine.IL-1 beta plays a role in angiogenesis by inducing VEGF production synergistically with TNF and IL6

Protein Expression

• IL-1 beta is expressed in activated monocytes/macrophages (at the protein level).
• Transcription and translation induced by M.tuberculosis and a number of different M.tuberculosis components in macrophages; EsxA is the most potent activator tested (at protein level). In pancreatic islets, release is increased by high glucose treatment. In pancreatic islets and macrophages, release is also increased by endocannabinoid anandamide/AEA。

Protein Localization

• Cytoplasm, lysosome, exosomes, secreted.
• The precursor is localized in the cytoplasm and, in response to inflammasome-activating signals such as ATP for the NLRP3 inflammasome or bacterial flagellin for the NLRC4 inflammasome, cleaved and secreted.

Figure 2: IL-1 beta Target ICC Experiment Results, Anti-IL-1 beta antibody [EPR23851-127] product (ab254360). Green: IL-1 beta, Red: alpha Tubulin, Blue: DAPI.

Isoforms & Post-translational Modifications

• Human (P01584): 30.7 kDa (predicted)
• Mouse (P10749): 30.9 kDa (predicted)
• Rat (Q63264): 30.6 kDa (predicted)
• The activation of the IL-1 beta precursor involves proteolysis catalyzed by CASP1. The processing and secretion are temporally correlated.

WB Experiment Tips

Precautions

• To increase IL-1 beta content in samples, it is recommended to pre-treat immune cells with TPA (12-O-tetradecanoylphorbol-13-acetate)/PMA (phorbol-12-myristate-13-acetate) (Figure 3). These are commonly used phorbol esters that bind and activate protein kinase C, facilitating intracellular signal transduction and cytokine production by various immune cells.
• IL-1 beta is not constitutively expressed, and both the precursor and mature forms may require stimulation to be detectable (Figures 4, 5). The induction conditions provided in product datasheets or literature are for reference only. When detecting IL-1 beta in different samples, optimize the concentration and duration of the inducer before formal testing to find the most suitable conditions. The intensity and type of stimulation affect IL-1 beta cleavage and secretion, and different products may require varying stimulation intensities. Refer to specific product datasheets, validate induction conditions using multiple products, or check the induction with another pathway target like TNF alpha to ensure appropriateness.
• As IL-1 beta is a secreted protein, it is recommended to treat cells with secretion inhibitors (e.g., Brefeldin A) to prevent IL-1 beta from being secreted extracellularly (Figures 3, 5).
• Ensure consistent cell culture density to maintain experimental success and reproducibility. For example, in THP-1 derived macrophages, due to quorum sensing, both low and high cell densities can affect cytokine secretion, including IL-1 beta.
• The precursor pro-IL-1 beta is approximately 31 kDa, and upon stimulation, the ~266 amino acid pro-IL-1 beta precursor is cleaved into the mature form of IL-1 beta with approximately 153 amino acids.
• IL-1 beta expression varies among different tissues and cells; some samples may show weak or no expression. Select appropriate experimental samples and use positive controls to confirm the experimental system is functioning correctly.
• IL-1 beta has low homology across species (e.g., human IL-1 beta has 67% homology with mouse IL-1 beta and 66% with rat IL-1 beta). Therefore, it is recommended to choose products with immunogens homologous to the samples being tested.

Positive Controls

• Human: THP-1 whole cell lysate pre-treated with TPA, followed by LPS and BFA treatment; U-87MG whole cell lysate
• Mouse: RAW 264.7 whole cell lysate treated with LPS and BFA

Negative Controls (No or Weak Expression)

• Human: Untreated THP-1 whole cell lysate
• Mouse: Untreated RAW 264.7 whole cell lysate

Example Results

Figure 3: WB - Anti-IL-1 beta antibody [EPR23851-127] (ab254360)

Lane 1 : Untreated RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 2 : RAW 264.7 treated with 100 ng/ml LPS for 7 hours and 300 ng/ml Brefeldin A (ab193369) for the last 3 hours, whole cell lysate
Lane 3 : Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate
Lane 4 : THP-1 treated with 80 nM TPA (ab120297) overnight and then 100 ng/ml LPS for 6 hours and 300 ng/ml Brefeldin A (ab193369) for the last 3 hours, whole cell lysate

Primary Antibody: Anti-IL-1 beta antibody [EPR23851-127] at 1/1000 dilution
Secondary Antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution
Exposure Time: 3 minutes
Predicted Band Size: 31 kDa
Observed Band Sizes: 17.5, 28, 31 kDa

Note: Expression of IL-1 beta is induced by LPS treatment. 31-kDa precursor IL-1 beta, 28- and 17.5-kDa proteolytically cleaved IL-1 beta are observed. The expesssion pattern and molecular weight observed is consistent with what has been described in the literature (PMID: 8446594, 19559631).

Figure 4: WB-Anti-IL-1 beta antibody [EPR21086] (ab216995)

Lane 1: 20 µg untreated THP-1 whole cell lysate
Lane 2: 20 µg THP-1 cell lysate treated with 100 ng/ml LPS for 3 hours

Primary antibody: Anti-IL-1 beta antibody [EPR21086] at 1/1000 dilution
Secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Exposure time: 3 minutes

Figure 5: WB- Anti-IL-1 beta antibody [EPR16805-15] (ab234437)

Lane 1: 10 µg untreated RAW 264.7 whole cell lysate
Lane 2: 10 µg RAW 264.7 whole cell lysate treated with 100 ng/ml LPS for 6 hours, then with 300 ng/ml Brefeldin A added aftert 3 hours

Primary antibody: Anti-IL-1 beta antibody [EPR16805-15] qt 1/1000 dilution
Secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 31 kDa
Detected band sizes: 17, 28, 31 kDa

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

1. Add a protease inhibitor cocktail to prevent degradation of target proteins.
2. Keep samples on ice throughout the entire sample preparation process.
3. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.
4. It is recommended to Select positive and negative controls.

Electrophoresis:

1. Load at least 20 μg of total protein for electrophoresis.
2. For target proteins with smaller molecular weights (e.g., <25 kDa), use a higher gel concentration for electrophoresis.

Transfer:

1. It is recommended to stain the membrane with Ponceau S after the transfer to confirm the success of the transfer.
2. It is advised not to cut membranes.

References

1. Bent R, Moll L, Grabbe S, Bros M. Interleukin-1 Beta-A Friend or Foe in Malignancies? Int J Mol Sci. 2018, 19(8):2155. doi: 10.3390/ijms19082155.
2. Kwak A; Lee Y; Kim H; Kim S. Intracellular interleukin (IL)-1 family cytokine processing enzyme. Arch. Pharm. Res. 2016, 39, 1556–1564. doi: 10.1007/s12272-016-0855-0.
3. Afonina IS; Muller C; Martin SJ.; Beyaert, R. Proteolytic processing of interleukin-1 family cytokines: Variations on a common theme. Immunity 2015, 42, 991–1004. doi: 10.1016/j.immuni.2015.06.003.
4. Andrei C, Margiocco P, Poggi A, Lotti LV, Torrisi MR, Rubartelli A. Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes. Proc. Natl. Acad. Sci. U.S.A. 2004, 101:9745-9750. doi: 10.1073/pnas.0308558101.
5. Netea MG, van de Veerdonk FL, van der Meer JW, Dinarello CA, Joosten LA: Inflammasome-independent regulation of IL-1-family cytokines. Annu Rev Immunol 2015. 33: 49–77. doi: 10.1146/annurev-immunol-032414-112306.
6. Lopez-Castejon G, Brough D. Understanding the mechanism of IL-1beta secretion. Cytokine Growth Factor Rev. 2011, 22(4):189-95. doi: 10.1016/j.cytogfr.2011.10.001.
7. Molineaux SM, Casano FJ, Rolando AM et al. Interleukin 1 beta (IL-1 beta) processing in murine macrophages requires a structurally conserved homologue of human IL-1 beta converting enzyme. Proc Natl Acad Sci U S A. 1993. 90(5):1809-13. doi: 10.1073/pnas.90.5.1809.
8. Adele V Ruder, Lieve Temmerman, Joep M A van Dommelen et al. Culture density influences the functional phenotype of human macrophages. Front Immunol. 2023 Mar 10:14:1078591. doi: 10.3389/fimmu.2023.1078591. eCollection 2023.